NOVEL INTERFERON-(lambda)4 (IFNL-4) PROTEIN, RELATED NUCLEIC ACID MOLECULES, AND USES THEREOF

ABSTRACT

The invention is related to identification of an interferon-analog (IFNL4) protein and genetic association with spontaneous clearance of HCV infection and response to treatment for HCV infection.

TECHNICAL FIELD

The present invention is related to identification of a novel humaninterferon, designated as interferon-λ4 (IFNL4) and methods of using itsmRNA, protein expression or protein activity to predict the clinicaloutcome of an HCV infection in an individual. It also relates to the useof the novel protein to identify novel compounds for treating an HCVinfection.

BACKGROUND OF INVENTION

Hepatitis C virus (HCV) is a single-stranded RNA virus in theFlaviviridae family of viruses. It is estimated that approximately 170million people worldwide, and at least 4 million people in the UnitedStates, have been infected with HCV (Thomas D L, Astemborski J, Rai R M,Anania F A, Schaeffer M, Galai N, Nolt K, Nelson K E, Strathdee S A,Johnson L, Laeyendecker O, Boitnott J, Wilson L E, Vlahov D., TheNatural History of Hepatitus C Virus Infection. JAMA 2000; 284 (4):450-456). In the US, more people die of HCV than HIV infection (Ly, K.,Xing J, Klevens R M, Jiles R B, Ward J W, Holmberg S D. The IncreasingBurden of Mortality From Viral Hepatitis in the United States Between1999 and 2007. Annals of Internal Medicine 156, 271-278 (2012).) Thus,infection with HCV represents a significant, worldwide health problem.

In most people, acute infection with HCV generally results in mildsymptoms such as fatigue, decreased appetite, and flu-like symptoms. Byconvention, acute hepatitis refers to the presence of clinical signs orsymptoms of hepatitis for a period of 6 months or fewer after thepresumed time of exposure. In some instances, however, the newlyinfected individual remains asymptomatic. While some individuals canspontaneously clear the virus, approximately 85% of people infected withHCV will develop chronic hepatitis C, which is defined as persistentviremia occurring at least 6 months after initial exposure (Blackard JT, Shata M T, Shire N J, Sherman K E., Acute Hepatitus C VirusInfection: A Chronic Problem., Hepatology 2008; 47(1):321-331). Chronicinfection with HCV is a leading cause of liver cancer and end-stageliver disease. It is also the most common reason for livertransplantation in the U.S. Currently, the standard treatment for HCVinfections is pegylated interferon-α (IFN-α) combined with ribavirin.Successful treatment resolves chronic HCV infection, thereby markedlyreducing HCV related morbidity and mortality, but the pegylatedIFN-α/ribavirin regimen is effective in less than 45% of patients, isexpensive and has many adverse effects. More recently, a triple therapycomprising pegylated-IFN-α, ribavirin, and an HCV protease inhibitor hasbeen recommended. Although this new regimen should be more efficaciousthan treatment with pegylated-interferon-α/ribavirin, a sizeableproportion of patients may fail to respond and patients treated withthis regimen will experience the adverse effects seen withpegylated-IFN-α/ribavirin therapy. Thus, a method of identifyingpatients who are unlikely to respond to treatment with interferon-basedtherapies is urgently desired so that these patients can be spared theexpense and adverse effects associated with futile treatment. Inaddition, the failure of some patients to respond to treatment indicatesthe need for new treatments for hepatitis C infections.

Increasing evidence suggests that host genetic factors influence boththe natural course of chronic HCV infection and response to therapy(Lauer G M, Walker B D. Hepatitis C virus infection. N Engl J Med 2001Jul. 5; 345(1):41-52; Manns M P, McHutchison J G, Gordon S C, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirincompared with interferon alfa-2b plus ribavirin for initial treatment ofchronic hepatitis C: a randomised trial. Lancet 2001 Sep. 22;358(9286):958-965; Fried M W, Shiffman M L, Reddy K R, Smith C, MarinosG, Goncales F L, Jr., et al. Peginterferon α-2a plus ribavirin forchronic hepatitis C virus infection. N Engl J Med 2002 Sep. 26;347(13):975-982; Kau A, Vermehren J, Sarrazin C. Treatment predictors ofa sustained virologic response in hepatitis B and C. J Hepatol 2008October; 49(4):634-651). For example, in two cohorts of pregnant womeninfected under similar conditions from immunoglobulin preparationscontaminated with a single strain of HCV, half spontaneously cleared theinfection and half progressed to chronic hepatitis C (Grakoui A, ShoukryN H, Woollard D J, Han J H, Hanson H L, Ghrayeb J, et al. HCVpersistence and immune evasion in the absence of memory T cell help.Science 2003 Oct. 24; 302(5645):659-662; Knapp S, Yee L J, Frodsham A J,Hennig B J, Hellier S, Zhang L, et al. Polymorphisms ininterferon-induced genes and the outcome of hepatitis C virus infection:roles of MxA, OAS-1 and PKR. Genes Immun 2003 September; 4(6):411-419).Among chronically infected patients, response to treatment differs, evenbetween cases with similar HCV-RNA levels and identical genotypes (ThioC L. Host genetic factors and antiviral immune responses to hepatitis Cvirus. Clin Liver Dis 2008 August; 12(3):713-26, xi.; Yee L J. Hostgenetic determinants in hepatitis C virus infection. Genes Immun 2004June; 5(4):237-245; Muller R. The natural history of hepatitis C:clinical experiences. J Hepatol 1996; 24(2 Suppl):52-54). The responserates are strongly associated with ethnicity (Conjeevaram, H. S. et al.Peginterferon and ribavirin treatment in African American and CaucasianAmerican patients with hepatitis C genotype 1. Gastroenterology,131:470-7 (2006)). Previous reports revealed the influence of geneticpolymorphisms of human leukocyte antigens (HLA) (Sheppard, P. et al.IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat Immunol 4,63-8 (2003); Robek, M. D., Boyd, B. S. & Chisari, F. V. Lambdainterferon inhibits hepatitis B and C virus replication. J. Virol. 79,3851-3854 (2005)), killer immunoglobulin-like receptors (KIRs)(Lauterbach, H. et al. Mouse CD8alpha+ DCs and human BDCA3+ DCs aremajor producers of IFN-lambda in response to Poly I:C. J Exp Med 207,2703-17), cytokines (WO 00/08215), chemokines and interleukins as wellas interferon-stimulated genes on HCV infection outcomes (Lasfar, A. etal. Characterization of the mouse IFN-lambda ligand-receptor system:IFN-lambdas exhibit antitumor activity against B16 melanoma. Cancer Res66, 4468-77 (2006); Phillips, J. E. & Corces, V. G. CTCF: master weaverof the genome. Cell 137, 1194-211 (2009); Shyu, A. B., Wilkinson, M. F.& van Hoof, A. Messenger RNA regulation: to translate or to degrade.EMBO J 27, 471-81 (2008); Conjeevaram, H. S. et al. Peginterferon andribavirin treatment in African American and Caucasian American patientswith hepatitis C genotype 1. Gastroenterology 131, 470-7 (2006); Ghany,M., Nelson, D. R., Strader, D. B., Thomas, D. L. & Seeff, L. B. Anupdate on treatment of genotype 1 chronic hepatitis c virus infection:2011 practice guidelines by the American association for the Study ofLiver Diseases. Hepatology, December 12 (doi: 10.1002/hep.25524) (2011).

Previous studies have used a candidate gene approach based on a prioriknowledge of the potential role of a gene in HCV infection. However,previous data do not allow accurate prediction of spontaneous clearanceor response to treatment (Robek, M. D., Boyd, B. S. & Chisari, F. V.Lambda interferon inhibits hepatitis B and C virus replication. J.Virol. 79, 3851-3854 (2005)). In 2009, several groups reported resultsfrom independent genome-wide association studies (GWAS) that identifiedsingle nucleotide polymorphisms (SNPs) in the IFNL3 (IL28B) gene regionthat are associated with response to pegylated IFN-α/ribavirin treatmentamong patients with chronic hepatitis C, as well as spontaneousclearance of HCV infection. For example, U.S. Patent Publication No.2011/0165124 by Bochud et al, which is incorporated herein in itsentirety by reference, discloses numerous SNPs associated with bothresponse to interferon-based treatment of HCV, and spontaneousclearance. Among the SNPs identified in these GWAS, the genotype basedon rs12979860 is currently accepted as the best predictor of spontaneousclearance and treatment response (Rauch, A. et al. Genetic variation inIL28B Is associated with chronic hepatitis C and treatment failure: agenome-wide association study. Gastroenterology 138, 1338-1345 (2010);Thomas, D. L. et al. Genetic variation in IL28B and spontaneousclearance of hepatitis C virus. Nature 461, 798-801 (2009); Ge, D. etal. Genetic variation in IL28B predicts hepatitis C treatment-inducedviral clearance. Nature 461, 399-401 (2009); Suppiah, V. et al. IL28B isassociated with response to chronic hepatitis C interferon-alpha andribavirin therapy. Nat Genet 41, 1100-4 (2009); Tanaka, Y. et al.Genome-wide association of IL28B with response to pegylatedinterferon-alpha and ribavirin therapy for chronic hepatitis C. NatGenet 41, 1105-9 (2009)). A single nucleotide polymorphism (SNP)rs12979860 is located approximately 3 kb upstream of the IFNL3 (IL28B)translational start site. Commercial laboratory tests based onrs12979860 are now available for predicting a patient's probability ofresponding to treatment.

Compared to persons of European ancestry, African American patients havea higher frequency of chronic hepatitis C and a poorer response totherapy with IFN-α/ribavirin. Racial differences in the frequency ofGWAS marker rs12979860 do not completely explain these disparities.Identification of a genetic marker that has optimal predictive values inall population groups would improve clinical decision models fortreatment of chronic hepatitis C and help deliver personalized medicineto all HCV-infected patients.

While current tests have proved to be useful in identifying respondersto treatment of chronic HCV infection, there remains a need for a morerobust and accurate test for predicting spontaneous clearance andresponse to treatment. Moreover, current tests require the isolation andgenotyping of nucleic acid molecules from an individual. Finally, asnoted above, there remains a percentage of the population who do notrespond to treatment for chronic HCV infection with current therapies.Thus, a need exists for novel methods and treatments for these patients.The present invention satisfies these needs and provides other benefitsas well.

SUMMARY OF INVENTION

The invention is related to identification of a novel human protein,interferon-λ4 (IFNL4), and related nucleic acid molecules (e.g., DNA,mRNA), and their relation to spontaneous clearance of HCV infection andresponse to treatment for HCV infection.

In one embodiment, the invention provides an isolated protein thatcomprises contiguous amino acids from an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.In specific embodiments, the isolated protein activates theJAK/STAT-signal transduction pathway. In specific embodiments, theisolated protein comprises at least about 30, at least about 40, atleast about 60, at least about 80, at least about 100, at least about110, at least about 140 contiguous amino acids from an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5and SEQ ID NO:8.

In one embodiment, the isolated protein comprises a sequence of at least50 contiguous amino acids, wherein the at least 50 contiguous amino acidsequence is at least 92% identical over its entire length to an at least50 contiguous amino acid sequence from an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

In one embodiment, the isolated protein comprises a sequence of at least150 contiguous amino acids that is at least 92% identical over itsentire length to an at least 150 contiguous amino acid sequence from SEQID NO:2.

In one embodiment, the isolated protein comprises a sequence of at least50 contiguous amino acids, wherein the at least 50 contiguous amino acidsequence is at least 92% identical over its entire length to an at least50 contiguous amino acid sequence from an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2 and further comprises at leastone sequence feature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises an amino acid sequenceat least 92% identical over its entire length to an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQID NO:8, wherein the isolated protein comprises at least one sequencefeature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises an amino acid sequenceat least 92% identical over its entire length to an SEQ ID NO:2, whereinthe isolated protein comprises at least one sequence feature selectedfrom the group consisting of:

-   -   a. an cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the protein possesses at least one activity selectedfrom the group consisting of: eliciting an antibody that selectivelybinds a protein consisting of SEQ ID NO:2, selectively binding anantibody generated against a protein consisting of SEQ ID NO:2,selectively binding a compound that binds to a protein consisting of SEQID NO:2, activating expression of the JAK/STAT pathway, and inducingexpression of at least one ISG listed in FIG. 15.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising a nucleic acid sequence selected from a nucleic acidsequence encoding an isolated protein of the present invention, and anucleic acid sequence fully complementary thereto. In certainembodiments the nucleic acid molecule comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9. One embodiment of theinvention provides a plasmid containing an isolated nucleic acidmolecule of the invention. Similarly, another embodiment of theinvention provides a virus containing an isolated nucleic acid moleculeof the invention.

One embodiment of the invention is an isolated antibody that selectivelybinds to an IFNL4 protein of the invention. Another embodiment of theinvention provides an isolated antibody that inhibits the binding of anantibody that selectively binds to an IFNL4 protein of the invention.

One embodiment of the invention is a method for predicting thelikelihood of an individual to spontaneously clear an HCV infection, byobtaining a biological sample from the individual; and analyzing thesample to determine the presence or absence of an IFNL4 mRNA or proteinof the present invention. In this method, the absence of IFNL4 mRNA orIFNL4 protein indicates an increased likelihood of the individualspontaneously clearing an HCV infection. In this method, the presence ofIFNL4 mRNA or protein indicates a decreased likelihood of the individualspontaneously clearing an HCV infection.

One embodiment of the invention is a method for predicting thelikelihood that an individual will respond to a treatment for HCVinfection by obtaining a biological sample from the individual andanalyzing the sample to determine the presence or absence of IFNL4 mRNAor IFNL4 protein of the present invention. In this method, the absenceof IFNL4 mRNA or IFNL4 protein of the present invention indicates anincreased likelihood the individual will respond to treatment for an HCVinfection. Alternatively, in this method the presence of IFNL4 mRNA orIFNL4 protein indicates a decreased likelihood the individual willrespond to treatment for an HCV infection. In one embodiment, thepresence of IFNL4 protein indicates the individual is predicted to beunable to respond to treatment for an HCV infection.

One embodiment of the invention is a method for predicting thelikelihood of an individual to spontaneously clear an HCV infection byobtaining a biological sample from an individual and determining thelevel of IFNL4 mRNA or IFNL4 protein present in the sample, if any. Inthis embodiment the level of IFNL4 mRNA or IFNL4 protein present in thesample indicates the likelihood the individual will spontaneously clearan HCV infection. In this method, a level of IFNL4 mRNA or IFNL4 in thesample less than the level of IFNL4 mRNA or IFNL4 protein present in asubject known to be able to clear an HCV infection indicates theindividual is predicted to be able to clear an HCV infection. In thismethod, a level of IFNL4 mRNA or IFNL4 in the sample greater than thelevel of IFNL4 mRNA or IFNL4 protein present in a subject known to beunable to clear an HCV infection indicates the individual is predictedto be unable to clear an HCV infection.

One embodiment of the invention is a method for treating a patientsuffering from a chronic hepatitis C virus infection by obtaining abiological sample from the individual, analyzing the sample to determinethe presence, absence or level of IFNL4 mRNA or IFNL4 protein present inthe sample and determining whether or not to administer treatment basedon the presence, absence or amount of IFNL4 mRNA or IFNL4 proteinpresent in the sample.

One embodiment of the invention is a kit useful for determining thepresence, absence or level of IFNL4 protein in a sample. The kitcomprises an antibody that specifically recognizes an IFNL4 protein ofthe invention. In one embodiment, the kit comprises instructions fordetermining the ability of an individual to spontaneously clear an HCVinfection. In one embodiment, the kit comprises instructions fordetermining the ability of an individual to respond to treatment for anHCV infection.

In one embodiment, the present invention provides a variant IFNL4polypeptide having at least 90%, at least 95%, at least 97%, at least98% or at least 99% sequence identity to a protein selected from thegroup consisting of IFNL4-p179, p131 and p107 (SEQ ID NO:2, SEQ ID NO:5and SEQ ID NO:8) wherein the variant polypeptide has at least one aminoacid substitution, using the numbering system of SEQ ID NO:2, selectedfrom the group consisting of A10P, A11P, L13M, V15F, C17Y, V19M, I20V,A21P, R26G, L28M, L35M, L40M, G116R, A121P, A126P, P128A, G129A, S130P,R132G, P135A, K139R, R140G, K143E, R146K, S149P, P150A, K154E, A155P,S156G, V1581, F159V, L162M, L164M, and L169F.

In one embodiment, the present invention provides a variant IFNL4polypeptide having at least 90%, at least 95%, at least 97%, at least98% or at least 99% sequence identity to a protein selected from thegroup consisting of IFNL4 p179, p131 and p107 (SEQ ID NO:2, SEQ ID NO:5and SEQ ID NO:8) wherein the variant polypeptide has at least one aminoacid substitution, using the numbering system of SEQ ID NO:2, selectedfrom selected form the group consisting of A10P, A11P, L13M, V15F, C17Y,V19M, 120V, A21P, R26G, L28M, L35M, L40M, E52K, L55M, W57R, R60P, N61H,S63P, F64V, R65G, D69H, P70S, P71T, R72G, G116R, A121P, A126P, P128A,G129A, S130P, R132G, P135A, K139R, R140G, K143E, R146K, S149P, P150A,K154E, A155P, S156G, V1581, F159V, L162M, L164M, and L169F.

In one embodiment, the present invention provides a variant IFNL4polypeptide having at least 90%, at least 95%, at least 97%, at least98% or at least 99% sequence identity to a protein selected from thegroup consisting of IFNL4-p179, p131 and p107 (SEQ ID NO:2, SEQ ID NO:5and SEQ ID NO:8) wherein the variant polypeptide has at least one aminoacid substitution, using the numbering system of SEQ ID NO:2, selectedfrom the group consisting of A10P, A11P, L13M, V15F, C17Y, V19M, 120V,A21P, R26G, L28M, L35M, L40M, E52K, L55M, W57R, R60P, N61H, S63P, F64V,R65G, D69H, P70S, P71T, R72G, R78G, V92M, L931, L101M, L102F, G116R,A121P, A126P, P128A, G129A, S130P, R132G, P135A, K139R, R140G, K143E,R146K, S149P, P150A, K154E, A155P, S156G, V1581, F159V, L162M, L164M,and L169F.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Results of RNA-sequencing (RNA-seq) in normal primary humanhepatocytes activated with 50 ug/ml of PolyI:C for 0, 1, 2, 4, 8, or 24hours. The results are presented for the 150 Kb genomic region of humanchromosome 19 that includes IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B)and several other genes used as unrelated controls. Location ofassociated genetic variants rs12979860 and rs8099917 is indicated.

FIG. 2. Identification by RNA-seq of a novel IFNL4 gene encodinginterferon-λ4 protein upstream of IFNL3 (IL28B) gene. The RNA-seq showstime-dependent activation of IFNL3 (IL28B) and a novel transcribedregion in primary human hepatocytes treated with PolyI:C.

FIG. 3. Splicing architecture of the ten novel transcripts (NCBIaccession numbers are presented in FIG. 4. The GWAS marker rs12979860 islocated within intron 1, and a novel marker, ss469415590, with TT or ΔGalleles is located within exon 1 and is common to all transcripts.Translation start site is marked by an arrow; ORFs are shaded in blue;aa, amino acids. The IFNL4-specific protein-coding frame is created by aΔG deletion allele of ss469415590 within the first exon of IFNL4 mRNAtranscripts. The IFNL4 protein isoforms of 107, 131 or 179 aa arecreated by transcripts with 3, 4 or 5 exons.

FIG. 4. Information on transcripts identified within the IFNL4 regionand submitted to NCBI GenBank.

FIG. 5. mRNA expression of full-length IFNL4 isoforms in humanhepatocyte samples homozygous for ΔG or TT alleles of a genetic variantss469415590 and activated with PolyI:C for 0, 2, 4 and 24 h. Thedeletion (ΔG) allele introduces a frame-shift that creates proteins of179, 131 or 107 aa. The insertion (TT) allele creates severalprematurely terminated transcripts that are likely to be degraded bynonsense-mediated decay. The primers detect the full-length amplicon ofIFNL4, including the start and stop codons. Also shown is expression ofIFNL3 (IL28B) and IFNL1 (IL29) transcripts, which are induced by PolyI:Ctreatment in samples with both genotypes. Expression of endogenouscontrol, PPIA, in the same samples is used as a loading control. Allsamples were treated with DNAse I and the signal should be specific forRNA expression only.

FIG. 6 Comparisons between IFNL4-p179 protein and selected members ofthe class-2 cytokine family.

FIG. 7. Amino acid sequence alignment of IFNL4 protein isoforms p179,p131 and p107 and human IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B) andIFN-α proteins. Shaded—identical amino acids; marked by arrows arepositions of cysteines involved in disulfide bonds in IFNL3 (IL28B)protein (C16-C115, C50-C148, C167-C174); positions of frame-shiftvariant ss469415590, amino acids important for interaction with IL-28R1(#27, 33, 34, 36, 37, 44, 53, 155, 158) and with IL-10R2(*97, *100) areindicated; numbering is based on mature IFNL3 (IL28B) protein (Q8IZI9),after removal of leader peptide (Gad H, Dellgren, C, et al. Inteferon-λ,is functionally an interferon but structurally related to theinterleukin-10 family, the Journal of Biological Chemistry, 2009).Helical protein structure is marked according to Trivella et al.Structure and function of interleukin-22 and other members of theinterleukin-10 family, Cell. Mol. Life Sci., 2010. Also indicated arepositions of IFNL4 genetic variants.

FIG. 8. Western blot protein detection by the mouse anti-IFNL4monoclonal antibody. Proteins used—purified recombinant IFNL4-p179 at 4concentrations, crude lysate or conditioned media from HepG2 cellstransiently transfected with the IFNL4-p179-Halo expression construct,and purified IFNL4-p107, IFN-α and IFNL3 (IL28B) proteins.

FIG. 9. Confocal imaging of IFNL4 expression with an anti-IFNL4 mousemonoclonal antibody or an anti-Halo antibody in HepG2 cells transientlytransfected with IFNL4-Halo expression construct; both antibodiessimilarly detected intracellular expression of IFNL4.

Confocal imaging of endogenously expressed IFNL4 in primary humanhepatocytes (PHH) from an individual heterozygous for ss469415590, theIFNL4 expression is induced by treatment with PolyI:C or in-vitroinfection with HCV.

FIG. 10. Confocal imaging in PHHs from carriers of different genotypesof ss469415590 (TT/TT, TT/ΔG or ΔG/ΔG) treated with 50 μg/ml of polyI:Cfor 0, 2, 4, 8 or 24 h. Red, IFNL4; green, cytoskeleton (α-tubulin),blue, nuclei. Intracellular IFNL4 expression is detected only in the PHHfrom carriers of risk genotypes (TT/AG or ΔG/ΔG).

FIGS. 11a & 11 b. Overview of the mouse and rabbit anti-IFNL4 monoclonalantibodies: location within protein and detection pattern of differentprotein isoforms after transient transfection of correspondingexpression constructs in HepG2 cells.

FIG. 12. Pathway Finder Analysis using luciferase reporter constructsrepresenting 45 human signaling pathways in HepG2 cells. The cells weretransiently transfected with expression constructs or an empty vector ortreated with 10 ng/ml recombinant purified IFN-α, IFNL3, IFNL4 or withPBS. All results represent the mean values of two independent biologicaltransfection and/or treatment replicates. Error bars, s.d. The rectanglemarks reporters (ISRE-Luc and IRF3-Luc) significantly induced bytreatment with IFN-α, IFNL3 and transient transfection with IFNL4construct.

FIG. 13. Luciferase activity after transfection with constructexpressing IFNL4, p131 or p107 and treatment with recombinant purifiedIFN-α or IFNL3 in the HepG2 cell line transiently cotransfected with theISRE-Luc reporter. The results are normalized to the activity seen aftertransfection with empty vector (mock) and represent the mean values ofeight biological replicates. Luciferase activity after transienttransfection with construct expressing IFNL4, p131 or p107 in the HepG2cell line stably expressing the ISRE-Luc reporter. The results arenormalized to the activity seen after transfection with empty vector(mock) and represent the mean values of 11 biological replicates. Testfor antiviral effects of the expression constructs for IFNL4, p131 andp107 transiently transfected into Huh7-Lunet cells stably expressing asubgenomic luciferase-expressing HCV replicon (HCV-Luc) compared to theeffect seen after transfection with empty vector (mock). Resultsrepresent the mean values of four biological replicates. Error bars,s.e.m.

FIG. 14. Protein blot analysis of STAT1 phosphorylated at Tyr701(pSTAT1) and STAT2 phosphorylated at Tyr689 (pSTAT2) in HepG2 cellstransiently transfected with constructs expressing the six proteinisoforms, including IFNL4-p179-Halo. All constructs are fused with theHalo tag and produce proteins detectable with an antibody for theHalo-tag; the rabbit monoclonal antibody to IFNL4 recognizes p179 aswell as the nonfunctional isoforms p131 and p107.

FIG. 15. Analysis of top canonical pathways and individual transcriptsactivated by transient overexpression of IFNL4 construct in HepG2 cells,based on global RNA-seq analysis.

Expression of selected ISGs in HepG2 cells in different conditions wasanalyzed in cells untreated, or transfected with empty vector (mock);IFNL4-p179, IFNL4-p131 or IFNL4-p107; or treated with 10 ng/ml of IFN-αor IFNL3 (IL28B) alone or after transfection with mock or IFNL4-p179.Expression of ISGs was analyzed by qRT-PCR with specific assays andnormalized to expression of four endogenous controls measured in thesame samples. The data is presented on log 2 scale—less negative valuesindicate higher expression. Error bars indicate mean values with 95%confidence intervals.

FIG. 16. Test for activation of the JAK/STAT pathway using ISRE-LucCignal reporter transiently transfected into HepG2, 293T and HeLa cells.Activation by transiently co-transfected IFNL4-p179, p107 and p131expression constructs or by recombinant purified IFN-α and IFNL3 (IL28B)proteins at indicated concentrations. Fold response is for comparison tomock-control (transfection with empty vector). Error bars indicate meanvalues of 4-8 independent biological replicates with 95% confidenceintervals.

FIG. 17. Analysis of 83 site-directed mutants of IFNL4. Protein sequenceof IFNL4 is aligned with IFNL3 and conserved amino acids are shaded;IFNL4 single-point mutations at specific positions are marked by aminoacid numbers (above) and types of changes (below) the sequence.Biological activity is defined as the ability to activate JAK/STATpathway measured as an induction of a transiently transfected ISRE-LucCignal reporter construct in HepG2 cells, with 4 biological replicatesfor each of the constructs. Results for all mutants are normalized toactivity of the WT-IFNL4.

FIG. 18. Biological activity of 83 IFNL4 mutants in specificcategories—cysteines residues (n=7) and non-cysteine residues conserved(n=38) or non-conserved (n=38) between IFNL3 and IFNL4. Mutations of anyof the 6 non-polymorphic cysteines eliminate the activity of IFNL4. Theexception is for a natural genetic variant Cys17Tyr which did not affectthe ability of IFNL4 to activate ISRE-Luc reporter. Significantly higher(p=0.005 for a two-sided non-paired T-test) proportion of residuesconserved between IFNL3 and IFNL4 suggests these residues are criticalfor retaining biological activity of IFNL4. Seven non-conservedIFNL4-specific residues were found of importance for biological activityfor IFNL4.

FIG. 19. Amino acid changes caused by genetic variants within IFNL4gene. Each of the amino acid changes belongs to a specific haplotypewhich carries the ΔG allele of the ss460415590 variant, thus, theseamino acid changes only exist when IFNL4 protein is produced. No aminoacid changes occur on haplotypes with the TT allele of the ss460415590,when IFNL4 is not produced.

FIG. 20. Principal components analysis (PCA) based on expression of 33transcripts involved in antiviral response and measured by qRT-PCR inHepG2 transiently transfected with specific allelic protein constructs(WT-IFNL4, Cys17Tyr, Arg60Pro and Pro70Ser), in three biologicalreplicates. The PCA plot shows that Pro70Ser mutant differs from boththe WT-IFNL4 protein and the group of Cys17Tyr and Arg60Pro mutantswhich cluster close to each other.

FIG. 21. Heatmap plot for transcripts with expression significantlyaffected by transient expression of IFNL4 allelic protein constructs(WT-IFNL4, Cys17Tyr, Arg60Pro and Pro70Ser) in HepG2 cells, based onresults of experiment presented on FIG. 20. Mutants Cys17Tyr andArg60Pro show similar effects on these transcripts, while Pro70Sershowed most difference with WT-IFNL4, causing lower expression of IL15,IL18, CTSB, FOS and SPP1 transcripts compared to cells transfected withWT-IFNL4, and higher expression of DAK, IRF7, DHX58 and APOBEC3Gtranscripts compared to cells transfected with WT-IFNL4. Color chartcorresponds to log 2 scale expression changes caused by mutants comparedto WT-IFNL4.

DESCRIPTION OF EMBODIMENTS

The present invention generally relates to a novel interferon genereferred to as IFNL4 and corresponding mRNA and protein generated inindividuals that carry at least one deletion (ΔG) allele of thess460415590 genetic variant. It also relates to methods of using IFNL4mRNA or protein to determine the probability that an individual willspontaneously clear an HCV infection, or will respond to therapeutictreatment of an HCV infection. More specifically, the present inventionrelates to the discovery that the amount of IFNL4 mRNA or IFNL4 proteinproduced by an individual correlates with the probability that theindividual will spontaneously clear an HCV infection, or will respond totreatment for an HCV infection. The present invention also relates tousing IFNL4 protein to identify compounds that can be used to treat anindividual with an HCV infection.

The present invention is an extension of the inventor's previous work,described in detail in U.S. Provisional Application No. 61/543,620, nowInternational Application No. PCT/US12/59048, filed Oct. 5, 2012, whichis incorporated herein by reference in its entirety. In their previouswork, the inventors discovered a novel compound polymorphism referred toas ss469415590 (NCBI reference number NC_000019.9:[g.39739154delT;g.39739155T>G]). The ss469415590 polymorphism consistsof two nucleotide variations that occur at positions 39,739,154 and39,739,155 on human chromosome 19, the coordinates being based on theFebruary 2009 human genome reference (GRch37/hg19). More specifically,the ss469415590 polymorphism consists of a single base deletionpolymorphism (T/Δ) and a single base substitution polymorphism (T/G),which are in complete linkage disequilibrium (r²=1.0). The inventorshave also discovered that, following treatment with PolyI:C, novel mRNAtranscripts are produced from this region (FIG. 1). Analysis of thesetranscripts resulted in the identification of a single transcriptionsite, followed by a protein translation start site, suggesting that anovel protein is produced from this region (FIG. 2). Moreover, deletionof the thymidine at position 39,739,154 causes a frame shift at aminoacid 22 of the putative protein, thereby altering the downstream readingframe (FIG. 3). Analysis of transcripts from this region showed thatsuch a frame shift results in production of 6 putative proteins,including 3 novel related proteins: a protein of 179 amino acids (hereinreferred to as IFNL4-p179), as well as two isoforms of IFNL4, with 131and 107 amino acids (p131 and p107, respectively) that differ byinclusion of alternative exons. In total, 10 transcripts were detectedin the IFNL4 region and deposited to NCBI GenBank (FIG. 4). Expressionof IFNL4 mRNA was detected only in PolyI:C-activated hepatocytes from anindividual homozygous for a risk ss469415590 ΔG allele but not from anindividual homozygous for a non-risk TT allele (FIG. 5). Analysis of theIFNL4-p179 sequence showed that the protein has strong similarity to thehuman IFN-λ, proteins, particularly to IFNL3 (IL28B), and some otherclass-2 cytokine family proteins (FIG. 6, FIG. 7).

Accordingly, one embodiment of the present invention is an isolatedprotein that comprises at least about 30 contiguous amino acids, atleast about 40 contiguous amino acids, at least about 50 contiguousamino acids, at least about 60 contiguous amino acids, at least about 70contiguous amino acids, at least about 80 contiguous amino acids, atleast about 90 contiguous amino acids, or at least about 100 contiguousamino acids from an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8. In oneembodiment, the isolated protein comprises at least about 110 contiguousamino acids, at least about 120 contiguous amino acids, or an at leastabout 130 contiguous amino acids from an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In oneembodiment, the isolated protein comprises at least about 140 contiguousamino acids or at least about 150 contiguous amino acids from SEQ IDNO:2. With regard to an amino acid sequence, as used herein, the term“about” means the number of contiguous amino acids can vary by up to 5%.Thus, about 40 contiguous amino acids means the isolated protein cancomprise between 38-42 contiguous amino acids. In one embodiment, theisolated protein comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

Before further embodiments are described, it should be appreciated that,unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs. Moreover, in order to aidthe reader, the following general definitions are supplied in order tofacilitate the understanding of the present invention.

It is to be noted that the term “a” “an” “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising,”“including,” and “having” can also be used interchangeably. Furthermore,the phrase “selected from the group consisting of” refers to one or moremembers of the group in the list that follows, including mixtures (i.e.combinations) of two or more members. As used herein, “at least one”means one or more. The term “comprise” is generally used in the sense of“including”, that is to say “permitting the presence of one or morefeatures or components”. It is to be further understood that wheredescriptions of various embodiments use the term comprising, thoseskilled in the art would understand that in some specific instances, anembodiment can be alternatively described using the phrase “consistingessentially of”.

As used herein, the terms isolated, isolating, purified, and the like,do not necessarily refer to the degree of purity of a cell or moleculeof the present invention. Such terms instead refer to cells or moleculesthat have been separated from their natural milieu or from components ofthe environment in which they are produced. For example, a naturallyoccurring cell or molecule (e.g., a DNA molecule, a protein, etc.)present in a living animal, including humans, is not isolated. However,the same cell, or molecule, separated from some or all of the coexistingmaterials in the animal, is considered isolated. As a further example,according to the present invention, protein molecules that are presentin a sample of blood obtained from an individual would be consideredisolated. It should be appreciated that protein molecules obtained fromsuch a blood sample using further purification steps would also bereferred to as isolated, in accordance with the notion that isolateddoes not refer to the degree of purity of the cells. Moreover, anisolated protein of the present invention can be obtained, for example,from its natural source (e.g., human), be produced using recombinant DNAtechnology, or be synthesized chemically.

It is understood by those skilled in the art that the sequence of aprotein may vary, or may be altered, with little or no affect on theactivity of that protein. According to the present invention, suchproteins are referred to as variants, allelic variants, mutants,isoforms, or homologues. Such variants can arise naturally as a resultof an individual carrying two different alleles that encode allelicvariants, or they can be constructed using techniques such as geneticengineering. With regard to the nomenclature of proteins and theirvariants, one form of the protein may arbitrarily be designated as thereference form (e.g., wild-type) and other forms designated as mutants,variants, isoforms or homologues. For example, if a particular allele,and thus its encoded protein, is associated with a particular phenotypiccharacteristic (e.g., the absence of a disease), or is found in themajority of a population, the encoded form of the protein may bereferred to as a “wild-type form”, while other forms may be referred toas variants, mutants, isoforms, or homologues. With regard to thepresent invention, a protein comprising the sequence of SEQ ID NO:2, SEQID NO:5 or SEQ ID NO:8 will be considered the wild-type (wt) protein.

Thus, one embodiment of the present invention is an IFNL4 proteinvariant. More specifically, one embodiment of the present invention isan isolated protein that comprises a sequence of at least 50 contiguousamino acids, wherein the at least 50 contiguous amino acid sequence isat least 92% identical, at least 94% identical, at least 96% identicalor at least 98% identical over its entire length to an at least 50contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:5 or SEQ IDNO:8. In a further embodiment, the isolated protein comprises a sequenceof at least 100 contiguous amino acids, wherein the at least 100contiguous amino acid sequence is at least 92% identical, at least 94%identical, at least 96% identical or at least 98% identical over itsentire length to an at least 100 contiguous amino acid sequence from SEQID NO:2, SEQ ID NO:5 or SEQ ID NO:8. In yet a further embodiment, theisolated protein comprises a sequence of at least 150 contiguous aminoacids, wherein the at least 150 contiguous amino acid sequence is atleast 92% identical, at least 94% identical, at least 96% identical orat least 98% identical over its entire length to an at least 150contiguous amino acid sequence from SEQ ID NO:2. In one embodiment, theisolated protein comprises an amino acid sequence at least 92%identical, at least 94% identical, at least 96% identical or at least98% identical over the entire length of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.Methods of determining the percent identity between two proteins, ornucleic acid molecules, are known to those skilled in the art.

With regard to such variants, any type of alteration in the amino acidsequence is permissible so long as the variant retains at least oneIFNL4 protein activity described herein. Examples of such variationsinclude, but are not limited to, amino acid deletions, amino acidinsertions, amino acid substitutions and combinations thereof. Forexample, it is well understood by those skilled in the art that one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), amino acids can often beremoved from the amino and/or carboxy terminal ends of a protein withoutsignificantly affecting the activity of that protein. Similarly, one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids can often beinserted into a protein without significantly affecting the activity ofthe protein.

As noted, isolated variant proteins of the present invention can alsocontain amino acid substitutions as compared to the wild-type IFNL4disclosed herein. Any amino acid substitution is permissible so long asthe activity of the protein is not significantly affected. In thisregard, it is appreciated in the art that amino acids can be classifiedinto groups based on their physical properties. Examples of such groupsinclude, but are not limited to, charged amino acids, uncharged aminoacids, polar uncharged amino acids, and hydrophobic amino acids.Preferred variants that contain substitutions are those in which anamino acid is substituted with an amino acid from the same group. Suchsubstitutions are referred to as conservative substitutions.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;2) neutral hydrophilic: Cys, Ser, Thr;3) acidic: Asp, Glu;4) basic: Asn, Gln, His, Lys, Arg;5) residues that influence chain orientation: Gly, Pro; and6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Inpreferred embodiments, such substituted residues may be introduced intohuman IFNL4 protein within regions non-homologous to IFN-α and IFN-λ,proteins,

In making amino acid changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein is generally understood in the art (Kyte et al., 1982, J. Mol.Biol. 157:105-31). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein. Thefollowing hydrophilicity values have been assigned to these amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); andtryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. One may alsoidentify epitopes from primary amino acid sequences on the basis ofhydrophilicity.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the IFNL4protein, or to increase or decrease the affinity of the IFNL4 proteinsdescribed herein. Exemplary amino acid substitutions are shown below inTable 1.

TABLE 1 Amino Acid Substitutions Original Amino Acid ExemplarySubstitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu CysSer, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu,Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, Asn Met Leu, Phe, IlePhe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr,Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala

Thus, in one embodiment of the present invention, the IFNL4 proteinvariant comprises at least one amino acid substitution, wherein thesubstitution is a conservative substitution. In one embodiment, theoriginal amino acid is substituted with an exemplary substitution shownin Table 1.

With regard to amino acid substitutions, it has previously beendiscussed that IFNL4 proteins of the present invention share up to 29%sequence identity (and 40% sequence similarity) with human IFN-λ,proteins. Moreover, it is understood by those skilled in the art, thatinterferon proteins have several amino acids that are conserved. Thepresently disclosed IFNL4 proteins contain many of these conserved aminoacids. Such amino acids are highlighted in FIG. 7. Amino acids 27, 33,34, 36, 37, 44, 53, 155 and 158 are important for IFNL3 (IL28B)interaction with its first receptor IFNLR1 (IL28R1), while amino acids97 and 100 are important for interaction with the second receptor,IL10R2 (Gad H, Dellgren, C, et al. Inteferon-λ, is functionally aninterferon but structurally related to the interleukin-10 family, theJournal of Biological Chemistry, 2009). Thus, in various embodiments,isolated variant proteins of the present invention contain at least one,or all, of these conserved interferon residues. Accordingly, oneembodiment of the present invention is an isolated protein thatcomprises a sequence of at least 50 contiguous amino acids, wherein theat least 50 contiguous amino acid sequence is at least 92% identical, atleast 94% identical, at least 96% identical or at least 98% identicalover its entire length to an at least 50 contiguous amino acid sequencefrom SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8, and wherein the at least50 contiguous amino acid sequence comprises at least one sequencefeature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        One embodiment, the isolated protein comprises at least 2, 3, 4,        5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24 or 25 of any of sequence elements a-y.

In one embodiment, the isolated protein comprises a sequence of at least50 contiguous amino acids, wherein the at least 50 contiguous amino acidsequence is at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to an atleast 50 contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:5 orSEQ ID NO:8, and wherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises a sequence of at least100 contiguous amino acids, wherein the at least 100 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 100 contiguous amino acid sequence from SEQ ID NO:2, SEQ IDNO:5 or SEQ ID NO:8, and wherein the at least 100 contiguous amino acidsequence comprises at least one sequence feature selected from the groupconsisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment, the isolated protein comprises at least 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24 or 25 of any of sequence elements a-y.

In one embodiment, the isolated protein comprises a sequence of at least100 contiguous amino acids, wherein the at least 100 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 100 contiguous amino acid sequence from SEQ ID NO:2, SEQ IDNO:5 or SEQ ID NO:8, and wherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises a sequence of at least130 contiguous amino acids, wherein the at least 130 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 130 contiguous amino acid sequence from SEQ ID NO:2 or SEQID NO:5, and wherein the at least 130 contiguous amino acid sequencecomprises at least one sequence feature selected from the groupconsisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   o. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   p. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   q. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   r. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   s. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   t. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   u. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   v. a leucine residue at the position corresponding to position        161 of SEQ ID    -   w. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   x. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   y. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   z. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   aa. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment, the isolated protein comprises at least 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26 or 27 of any of sequence elements a-aa.

In one embodiment, the isolated protein comprises a sequence of at least130 contiguous amino acids, wherein the at least 130 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 130 contiguous amino acid sequence from SEQ ID NO:2 or SEQID NO:5, and wherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID    -   n. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   o. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   p. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   q. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   r. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   s. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   t. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   u. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   v. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   w. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   x. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   y. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   z. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   aa. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises a sequence of at least150 contiguous amino acids, wherein the at least 150 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 150 contiguous amino acid sequence from SEQ ID NO:2, andwherein the at least 150 contiguous amino acid sequence comprises atleast one sequence feature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment the isolated protein comprises at least 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28 or 29 of any of sequence elements        a-cc.

In one embodiment, the isolated protein comprises a sequence of at least150 contiguous amino acids, wherein the at least 150 contiguous aminoacid sequence is at least 92% identical, at least 94% identical, atleast 96% identical or at least 98% identical over its entire length toan at least 150 contiguous amino acid sequence from SEQ ID NO:2, andwherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

One embodiment of the present invention is an isolated proteincomprising an amino acid sequence at least 85% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to SEQ IDNO:8, wherein the isolated protein comprises at least one sequencefeature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment, the isolated protein comprises at least 2, 3,        4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24 or 25 of any of sequence elements a-y.

One embodiment of the present invention is an isolated proteincomprising an amino acid sequence at least 85% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to SEQ IDNO:8, wherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   m. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   n. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   p. a cysteine residue at the position corresponding to position        122 of SEQ ID    -   q. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   r. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   s. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   t. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   u. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   v. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   w. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   x. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   y. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

One embodiment of the present invention is an isolated proteincomprising an amino acid sequence at least 85% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to SEQ IDNO:5, wherein the isolated protein comprises at least one sequencefeature selected from the group consisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   o. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   p. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   q. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   r. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   s. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   t. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   u. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   v. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   w. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   x. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   y. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   z. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   aa. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment the isolated protein comprises at least 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26 or 27 of any of sequence elements a-aa.

One embodiment of the present invention is an isolated proteincomprising an amino acid sequence at least 85% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to SEQ IDNO:5, wherein the isolated protein comprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   o. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   p. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   q. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   r. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   s. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   t. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   u. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   v. a leucine residue at the position corresponding to position        161 of SEQ ID    -   w. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   x. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   y. a threonine residue at the position corresponding to position        166 of SEQ ID NO:2;    -   z. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   aa. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

In one embodiment, the isolated protein comprises an amino acid sequenceat least 85% identical, at least 90% identical, at least 92% identical,at least 94% identical, at least 96% identical or at least 98% identicalover its entire length to SEQ ID NO:2, wherein the isolated proteincomprises at least one sequence feature selected from the groupconsisting of:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID NO:2;    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.        In one embodiment the isolated protein comprises at least 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28 or 29 of any of sequence elements        a-cc.

In one embodiment, the isolated protein comprises an amino acid sequenceat least 85% identical, at least 90% identical, at least 92% identical,at least 94% identical, at least 96% identical or at least 98% identicalover its entire length to SEQ ID NO:2, wherein the isolated proteincomprises:

-   -   a. a cysteine residue at the position corresponding to position        27 of SEQ ID NO:2;    -   b. an leucine residue at the position corresponding to position        29 of SEQ ID NO:2    -   c. a serine residue at the position corresponding to position 30        of SEQ ID NO:2    -   d. a tyrosine residue at the position corresponding to position        32 of SEQ ID NO:2    -   e. a serine residue at the position corresponding to position 34        of SEQ ID NO:2    -   f. a proline residue at the position corresponding to position        37 of SEQ ID NO:2;    -   g. a leucine residue at the position corresponding to position        40 of SEQ ID NO:2;    -   h. an alanine residue at the position corresponding to position        42 of SEQ ID NO:2;    -   i. a lysine residue at the position corresponding to position 44        of SEQ ID NO:2;    -   j. an aspartic acid residue at the position corresponding to        position 48 of SEQ ID NO:2;    -   k. a tyrosine residue at the position corresponding to position        50 of SEQ ID NO:2;    -   l. a glutamic acid residue at the position corresponding to        position 51 of SEQ ID NO:2;    -   m. a cysteine residue at the position corresponding to position        62 of SEQ ID NO:2;    -   n. a cysteine residue at the position corresponding to position        76 of SEQ ID NO:2;    -   o. an alanine residue at the position corresponding to position        87 of SEQ ID NO:2;    -   p. a leucine residue at the position corresponding to position        111 of SEQ ID NO:2;    -   q. a leucine residue at the position corresponding to position        112 of SEQ ID NO:2;    -   r. an aspartic acid residue at the position corresponding to        position 118 of SEQ ID NO:2;    -   s. an alanine residue at the position corresponding to position        120 of SEQ ID NO:2;    -   t. a cysteine residue at the position corresponding to position        122 of SEQ ID NO:2;    -   u. a cysteine residue at the position corresponding to position        152 of SEQ ID NO:2;    -   v. a valine residue at the position corresponding to position        157 of SEQ ID NO:2;    -   w. an asparagine residue at the position corresponding to        position 160 of SEQ ID    -   x. a leucine residue at the position corresponding to position        161 of SEQ ID NO:2;    -   y. an arginine residue at the position corresponding to position        163 of SEQ ID NO:2    -   z. a leucine residue at the position corresponding to position        165 of SEQ ID NO:2;    -   aa. a threonine residue at the position corresponding to        position 166 of SEQ ID NO:2;    -   bb. an alanine residue at the position corresponding to position        173 of SEQ ID NO:2; and    -   cc. a cysteine residue at the position corresponding to position        178 of SEQ ID NO:2.

As noted above, IFNL4 proteins of the present invention have particularactivities that make them useful tools for diagnosing, and developingtreatments for individuals infected with HCV and individuals at risk forsuch infection. Examples of such activity include:

1) the ability to elicit an antibody that selectively binds a proteinconsisting of SEQ ID NO:2;

2) selectively binding an antibody generated against a proteinconsisting of SEQ ID NO:2;

3) selectively binding a compound that binds to a protein consisting ofSEQ ID NO:2, and;

4) the ability of IFNL4 to induce the JAK/STAT pathway.

Methods of measuring induction of the JAK/STAT pathway are known tothose skilled in the art. One such method is an assay based on theability of IFNL4 protein to stimulate expression of interferonstimulated genes (ISGs).

In one embodiment, an isolated protein of the present invention is ableto elicit an antibody that selectively binds to a protein consisting ofSEQ ID NO:2. In one embodiment, a specific mouse anti-IFNL4 antibody wasraised to recognize a synthetic peptide corresponding to amino acids44-74 of human IFNL4. The specific detection by Western blot wasdemonstrated using recombinant purified IFNL4 protein (FIG. 8) andconfocal imaging of HepG2 cells transiently transfected with IFNL4-Haloconstruct, and primary human hepatocytes from an individual heterozygousfor ss469415590 activated by treatment with PolyI:C or by in-vitroinfection of HCV (FIG. 9) Thus, embodiments of the invention includeantibodies that specifically recognize and bind to the IFNL4 protein, orfragments thereof (FIG. 11). In a specific embodiment, the antibodiesmay be monoclonal antibodies, raised in the mouse or rabbit.

In one embodiment, an isolated protein of the present invention is ableto bind a compound that binds to a protein consisting of SEQ ID NO:2. Inone embodiment, an isolated protein of the present invention is able toinduce the JAK/STAT pathway.

As used herein, the phrase without significantly affecting the activityof a protein means that the variant has at least 80%, at least 90% or atleast 95% of the activity of a protein consisting of SEQ ID NO:2. Thus,in one embodiment, the isolated protein has at least 80%, at least 90%or at least 95% of the activity of a protein consisting of SEQ ID NO:2.In one embodiment, the isolated protein has at least 98% of the activityof a protein consisting of SEQ ID NO:2. In some cases, variations in theamino acid sequence of the isolated protein will increase the activityof the protein. Thus, one embodiment of the present invention is anisolated protein that has at least 1.25×, at least 1.5×, at least 1.75×,at last 2×, at last 4×, at least 6×, at least 8× or at least 10× thelevel of activity of a protein consisting of SEQ ID NO:2. Themeasurement and comparison of such activities can be accomplished usingmethods known in the art. For example, the ability to elicit an antibodyto a protein is generally accomplished by immunizing an animal (e.g., amouse) with an isolated protein of the present invention, and thentesting the resulting antibodies for their ability to selectively bind aprotein, such as a protein consisting of SEQ ID NO:2. As used herein,the terms selectively, selective, specific, and the like, indicate theantibody has a greater affinity for an isolated IFNL4 protein than itdoes for proteins unrelated to IFNL4. More specifically, the termsselectively, selective, specific, and the like indicate that theaffinity of the antibody for an isolated IFNL4 protein is statisticallysignificantly higher than its affinity for a negative control (e.g., anunrelated protein, such as, for example, albumin) as measured using astandard assay (e.g., ELISA). Likewise, similar techniques, known tothose skilled in the art, exist for measuring the ability of a compoundto bind IFNL4 proteins of the present invention.

In addition to testing for the ability to elicit an antibody, IFNL4proteins of the present invention can be tested for their ability toactivate the JAK/STAT pathway. Such assays are generally performed bymeasuring the activity of components of the JAK/STAT system. Forexample, luciferase assays may be used to measure activity of a reportergene that serves as a measure of JAK/STAT activity. In these assays, theluciferase protein is used as a reporter gene under regulation of aninterferon-stimulated responsive element (ISRE) Among 45 human signalingpathways tested, only the ISRE-Luc and IRF3-Luc reporters were activatedby type I and III interferons (IFN-α and IFNL3) and a transientlytransfected expression construct for IFNL4, but not purified recombinantIFNL4 protein or expression constructs for other protein isoforms (FIG.12). This was validated by an individual ISRE-Luc assay in HepG2 cellstransiently or stably transfected with ISRE-Luc (FIG. 13). IFNL4 alsoshowed anti-viral response in a specific experiment in a Huh7 cell lineexpressing Luciferase reporter linked with a subgenomic HCV replicon(FIG. 13). In another embodiment, protein activity (measured for exampleby Western blot) of JAK/STAT pathway protein components may be measured.For example, tyrosine phosphorylation of STAT1 protein may be measuredby Western blot as an indicator of the activity of the cytokine receptorsignaling pathways. In another embodiment, the activity of the JAK/STATpathway may be measured by the expression level of interferon stimulatedgenes, ISGs, i.e., measurement of gene expression levels of genesdirectly or indirectly stimulated by interferons, resulting inactivation of the JAK/STAT pathway. Only transiently transfected IFNL4,but not other protein isoforms, caused STAT1 and STAT2 phosphorylation,similar to that caused by IFNL3 (FIG. 14).

Suitable techniques for assaying for the IFNL4 protein biologicalactivities are disclosed herein and are known to those skilled in theart. Such assays can be in vitro or in vivo assays. Examples of usefulassays include, but are not limited to, an enzyme-linked immunoassay, acompetitive enzyme-linked immunoassay, a radioimmunoassay, afluorescence immunoassay, a chemiluminescent assay, a lateral flowassay, a flow-through assay, an agglutination assay, a particulate-basedassay (e.g., using particulates such as, but not limited to, magneticparticles or plastic polymers, such as latex or polystyrene beads), animmunoprecipitation assay, an immunoblot assay (e.g., a western blot), aphosphorescence assay, a flow-through assay, a chromatography assay, apolyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmonresonance assay, a spectrophotometric assay, a particulate-based assay,an electronic sensory assay and a flow cytometric assay. Methods ofperforming such assays are well known to those skilled in the art.Assays can be designed to give qualitative, quantitative orsemi-quantitative results, depending on how they are used and the typeof result that is desired.

While isolated proteins of the present invention can consist entirely ofthe sequences disclosed herein, and the disclosed variants thereof, suchproteins may additionally contain amino acid sequences that do notconfer IFNL4 activity, but which have other useful functions. Anyuseful, additional amino acid sequence can be added to the isolatedprotein sequence, so long as the additional sequences do not have anunwanted effect on the protein's ability to elicit an antibody to aprotein consisting of SEQ ID NO:2, selectively bind an antibody thatselectively binds to a protein consisting of SEQ ID NO:2, bind acompound that binds to a protein consisting of SEQ ID NO:2, or activateexpression of the JAK/STAT pathway. For example, isolated proteins ofthe present invention can contain amino acid sequences that are usefulfor visualizing or purifying the peptide. Such sequences act as labels(e.g., enzymes) or tags (antibody binding sites). Examples of suchlabels and tags include, but are not limited to, β-galacosidase,luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotintags, and fluorescent tags. Other useful sequences for labeling andtagging proteins are known to those of skill in the art.

In addition to the modifications described above, isolated proteins ofthe present invention can be further modified, so long as suchmodification does not significantly affect the ability of the protein toelicit an antibody to a protein consisting of SEQ ID NO:2, selectivelybind an antibody that selectively binds to a protein consisting of SEQID NO:2, bind a compound that binds to a protein consisting of SEQ IDNO:2, or activate expression of the JAK/STAT pathway. Such modificationscan be made, for example, to increase the stability, solubility orabsorbability of the protein. Examples of such modifications include,but are not limited to pegylation, glycosylation, phosphorylation,acetylation, myristylation, palmitoylation, amidation and/or otherchemical modification of the peptide.

Isolated proteins of the present invention can be obtained from nature(e.g., obtained from plants, animals or microorganisms) or they can beproduced in a laboratory (e.g., recombinantly or synthetically). Alsoencompassed are peptides that are combinations of natural and syntheticmolecules. General methods for producing and isolating recombinant orsynthetic peptides are known to those skilled in the art.

Proteins of the present invention are encoded by isolated nucleic acidmolecules of the present invention. In accordance with the presentinvention, an isolated nucleic acid molecule is one that has beenremoved from its natural milieu (i.e., that has been subject to humanmanipulation). As such, isolated does not reflect the extent to whichthe nucleic acid molecule has been purified. An isolated nucleic acidmolecule of the present invention can be obtained from its naturalsource either as an entire (i.e., complete) gene, or as some portionthereof that encodes an isolated protein of the present invention.Alternatively, an isolated nucleic acid molecule of the presentinvention can be obtained through synthesis (e.g., chemical synthesis,solid-phase synthesis, polymerase chain reaction). Further, isolatednucleic acid molecules of the present invention can be a combination ofmolecules obtained from a natural source, and molecules obtained throughsynthesis (e.g., cloning of genome fragments encoding an isolatedprotein of the present invention). In addition, an isolated nucleic acidmolecule of the present invention can include DNA, RNA or derivativesthereof.

An isolated nucleic acid molecule of the present invention can beproduced using a number of methods known to those skilled in the art(see, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Edition, 2001, which is incorporated herein by referencein its entirety). For example, nucleic acid molecules can be modifiedusing a variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to “build” a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule variants can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., the ability of theprotein to ability to elicit an antibody to a protein consisting of SEQID NO:2, bind a compound that binds to a protein consisting of SEQ IDNO:2, or activate expression of the JAK/STAT pathway). Such screeningmethods have been described herein and are routinely performed by thoseskilled in the art.

Thus, one embodiment of the present invention is a nucleic acid moleculecomprising a nucleic acid sequence that encodes an isolated proteincomprising at least about 30 contiguous amino acids, at least about 40contiguous amino acids, at least about 50 contiguous amino acids, atleast about 60 contiguous amino acids, at least about 70 contiguousamino acids, at least about 80 contiguous amino acids, at least about 90contiguous amino acids, or at least about 100 contiguous amino acidsfrom an amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:5 and SEQ ID NO:8. In one embodiment, the isolatednucleic acid molecule comprise a nucleic acid sequence that encodes anisolated protein comprising at least about 110 contiguous amino acids,at least about 120 contiguous amino acids, or an at least about 130contiguous amino acids from an amino acid selected from the groupconsisting of SEQ IDNO:2 and SEQ ID NO:5. In one embodiment, theisolated nucleic acid molecule comprise a nucleic acid sequence thatencodes an isolated protein comprising at least about 140 contiguousamino acids or at least about 150 contiguous amino acids from SEQ IDNO:2. In one embodiment, the isolated nucleic acid molecule comprises atleast 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500contiguous nucleotides from a nucleic acid sequence selected form thegroup consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7. Alsoencompassed are nucleic acid molecules comprising nucleic acid sequencesfully complementary to nucleic acid sequences present in nucleic acidmolecules of the present invention. Thus, one embodiment of the presentinvention is an isolated nucleic acid molecule comprising at least 90,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 contiguousnucleotides from a nucleic acid sequence selected form the groupconsisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8.

As has been discussed, the present invention encompasses variants ofproteins comprising the sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ IDNO:8. Thus, one embodiment of the present invention is an isolatednucleic acid molecule that encodes a protein comprising a sequence of atleast 50 contiguous amino acids, wherein the at least 50 contiguousamino acid sequence is at least 92% identical, at least 94% identical,at least 96% identical or at least 98% identical over its entire lengthto an at least 50 contiguous amino acid sequence from SEQ ID NO:2, SEQID NO:5 or SEQ ID NO:8. In a further embodiment, the isolated nucleicacid molecule encodes a protein comprising a sequence of at least 100contiguous amino acids, wherein the at least 100 contiguous amino acidsequence is at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to an atleast 100 contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:5or SEQ ID NO:8. In yet a further embodiment, the isolated nucleic acidmolecule encodes a protein comprising a sequence of at least 150contiguous amino acids, wherein the at least 150 contiguous amino acidsequence is at least 92% identical, at least 94% identical, at least 96%identical or at least 98% identical over its entire length to an atleast 150 contiguous amino acid sequence from SEQ ID NO:2. In oneembodiment, the isolated nucleic acid molecule encodes a proteincomprising an amino acid sequence at least 92% identical, at least 94%identical, at least 96% identical or at least 98% identical over theentire length of an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8. In oneembodiment, the isolated nucleic acid molecule comprises at least 90, atleast 100, at least 125, at least 150, at least 175, at least 200, atleast 250, at least 300, at least 350, at least 400, at least 450, or atleast 500 contiguous nucleotides that are at least 90%, at least 92%, atleast 94%, at least 96% or at least 98% identical over their entirelength to at least 90, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450, or at least 500 contiguous nucleotides from anucleic acid sequence selected form the group consisting of SEQ ID NO:1,SEQ ID NO:4 and SEQ ID NO:7. In various embodiments, the isolatednucleic acid molecules encode proteins comprising the conserved aminoacids disclosed herein.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of anIFNL4 protein, or variants thereof. A preferred oligonucleotide iscapable of hybridizing, under stringent conditions, with a nucleic acidmolecule that is capable of encoding a protein comprising at least 30contiguous amino acids from SEQ ID NO:2, or a variant protein thereof.Certain preferred oligonucleotides are capable of hybridizing to nucleicacid molecules comprising at least 90, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, or 500 contiguous nucleotides from a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4and SEQ ID NO:7, or complements thereof. Preferred oligonucleotides arethose having at least 92%, at least 94%, at least 96% or at least 98%identity over their entire length with at least a portion of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimal size of such oligonucleotides is thesize required to form a stable hybrid between a given oligonucleotideand the complementary sequence on another nucleic acid molecule of thepresent invention. The size of the oligonucleotide must also besufficient for the use of the oligonucleotide in accordance with thepresent invention. Oligonucleotides of the present invention can be usedin a variety of applications including, but not limited to, as probes toidentify additional nucleic acid molecules, as primers to amplify orextend nucleic acid molecules or in therapeutic applications to inhibit,for example, expression of IFNL4 protein. Such therapeutic applicationsinclude the use of such oligonucleotides in, for example, antisense,triplex formation, ribozyme and/or RNA drug-based technologies. Suchtechnologies are known to those skilled in the art. The presentinvention, therefore, includes such oligonucleotides and methods tointerfere with the production of IFNL4 proteins by use of one or more ofsuch technologies.

The present invention also includes recombinant constructs, whichcomprise nucleic acid molecule of the present invention inserted intoany vector capable of delivering the nucleic acid molecule into a hostcell. Such a construct contains heterologous nucleic acid sequences,that is nucleic acid sequences that are not naturally found adjacent toIFNL4 protein encoding nucleic acid molecules of the present invention.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulating of IFNL4protein and/or IFNL4 nucleic acid molecules of the present invention.One type of recombinant construct herein referred to as a recombinantmolecule and described in more detail below, can be used in theexpression of nucleic acid molecules of the present invention. Preferredrecombinant vectors are capable of replicating in the transformed celllines.

A preferred nucleic acid molecule to include in a recombinant vector ofthe present invention is a nucleic acid molecule that encodes at least aportion of at least one IFNL4 protein of the present invention, orvariants thereof. A specific nucleic acid molecule to include in arecombinant vector is a nucleic acid molecules encoding at least about30 contiguous amino acids, at least about 40 contiguous amino acids, atleast about 50 contiguous amino acids, at least about 60 contiguousamino acids, at least about 70 contiguous amino acids, at least about 80contiguous amino acids, at least about 90 contiguous amino acids, or atleast about 100 contiguous amino acids from an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQID NO:8, or variants thereof. As such, also included are nucleic acidmolecules comprising at least 90, 100, 125, 150, 175, 200, 250, 300,350, 400, 450, or 500 contiguous nucleotides from a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4and SEQ ID NO:7, complements thereof, and variants thereof.

In one embodiment, an IFNL4 protein of the present invention is producedby culturing a cell capable of expressing the protein under conditionseffective to produce the protein, and recovering the protein. Apreferred cell to culture is a recombinant cell that is capable ofexpressing the IFNL4 protein, the recombinant cell being produced bytransforming a host cell with one or more nucleic acid molecules of thepresent invention. Transformation of a nucleic acid molecule into a cellcan be accomplished by any method by which a nucleic acid molecule canbe inserted into the cell. Transformation techniques include, but arenot limited to, transfection, electroporation, microinjection,lipofection, adsorption, and protoplast fusion. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Nucleic acidmolecules with which to transform a host cell are as disclosed hereinfor including in recombinant vectors of the present invention.

Suitable host cells to transform include any cell that can betransformed and that can express the introduced IFNL4 protein. Suchcells are, therefore, capable of producing IFNL4 proteins of the presentinvention after being transformed with at least one nucleic acidmolecule of the present invention. Host cells can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Suitable host cells of the present inventioncan include bacterial, fungal (including yeast), insect, animal andplant cells. Preferred host cells include bacterial, yeast, insect andmammalian cells, with bacterial (e.g., E. coli) and insect (e.g.,Spodoptera) cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase “operatively linked” refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth, insect and mammalian cells, such as, butnot limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB,bacteriophage lambda (λ) (such as λp_(L) and λp_(R) and fusions thatinclude such promoters), bacteriophage T7, T7 lac, bacteriophage T3,bacteriophage SP6, bacteriophage SP01, metallothionein, alpha matingfactor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such asSindbis virus subgenomic promoters), baculovirus, Heliothis zea insectvirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus40, retrovirus actin, retroviral long terminal repeat, Rous sarcomavirus, heat shock, phosphate and nitrate transcription control sequencesas well as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins). Transcription control sequences of thepresent invention can also include naturally occurring transcriptioncontrol sequences naturally associated with a DNA sequence encoding anIFNL4 protein.

Expression vectors of the present invention may also contain secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed protein to be secreted from the cell, or any heterologoussignal segment capable of directing the secretion of an IFNL4 protein,including fusion proteins, of the present invention. Preferred signalsegments include, but are not limited to, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of a nucleic acid molecule of the presentinvention can enhance the stability during production, storage and/oruse of the protein encoded by the nucleic acid molecule. Furthermore, afusion segment can function as a tool to simplify purification of anIFNL4 protein, such as to enable purification of the resultant fusionprotein using affinity chromatography. A suitable fusion segment can bea domain of any size that has the desired function (e.g., increasedstability and/or purification tool). It is within the scope of thepresent invention to use one or more fusion segments. Fusion segmentscan be joined to amino and/or carboxyl termini of an IFNL4 protein.Linkages between fusion segments and IFNL4 proteins can be constructedto be susceptible to cleavage to enable straight-forward recovery of theIFNL4 proteins. Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of an IFNL4 protein.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed. A preferred recombinantmolecule includes one or more nucleic acid molecules that encode atleast about 30 contiguous amino acids, at least about 40 contiguousamino acids, at least about 50 contiguous amino acids, at least about 60contiguous amino acids, at least about 70 contiguous amino acids, atleast about 80 contiguous amino acids, at least about 90 contiguousamino acids, or at least about 100 contiguous amino acids from an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:5 and SEQ ID NO:8, or variants thereof. Particularly preferrednucleic acid molecules to include in a recombinant molecule are asdisclosed herein for including in a recombinant vector of the presentinvention.

A recombinant cell of the present invention includes any cellstransformed with at least one of any nucleic acid molecules of thepresent invention. A preferred recombinant cell is a cell transformedwith at least one nucleic acid molecule that encodes at least about 30contiguous amino acids, at least about 40 contiguous amino acids, atleast about 50 contiguous amino acids, at least about 60 contiguousamino acids, at least about 70 contiguous amino acids, at least about 80contiguous amino acids, at least about 90 contiguous amino acids, or atleast about 100 contiguous amino acids from an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQID NO:8, or variants thereof.

It will be appreciated by those skilled in the art that use ofrecombinant DNA technologies can improve expression of transformednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within a host cell, the efficiencywith which those nucleic acid molecules are transcribed, the efficiencywith which the resultant transcripts are translated, and the efficiencyof post-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

In accordance with the present invention, recombinant cells can be usedto produce an IFNL4 protein of the present invention by culturing suchcells under conditions effective to produce such a protein, andrecovering the protein. Effective conditions to produce a proteininclude, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing anIFNL4 protein. Such a medium is typically an aqueous medium comprisingassimilable carbohydrate, nitrogen and phosphate sources, as well asappropriate salts, minerals, metals and other nutrients, such asvitamins. The medium may comprise complex nutrients or may be a definedminimal medium.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantIFNL4 proteins may either remain within the recombinant cell; besecreted into the fermentation medium; be secreted into a space betweentwo cellular membranes, such as the periplasmic space in E. coli; or beretained on the outer surface of a cell or viral membrane. The phrase“recovering the protein” refers simply to collecting the wholefermentation medium containing the protein and need not imply additionalsteps of separation or purification. IFNL4 proteins of the presentinvention can be purified using a variety of standard proteinpurification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, chromatofocusing anddifferential solubilization.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to isolated IFNL4proteins of the present invention and/or IFNL4 proteins present in anindividual or a sample from an individual. Isolated antibodies of thepresent invention can include antibodies in serum, or antibodies thathave been purified to varying degrees. Antibodies of the presentinvention can be polyclonal or monoclonal, or can be functionalequivalents such as antibody fragments and genetically-engineeredantibodies, including single chain antibodies or chimeric antibodiesthat can bind to one or more epitopes on IFNL4 proteins of the presentinvention. A suitable method to produce antibodies effective for use inthe present invention includes (a) administering to an animal aneffective amount of an IFNL4 protein, or fragment thereof, to producethe antibodies and (b) recovering the antibodies. Antibodies raisedagainst defined proteins or fragments can be advantageous because suchantibodies are not substantially contaminated with antibodies againstother substances that might otherwise cause interference in a diagnosticassay. Methods to produce such antibodies are known in the art and aredescribed in detail in Harlow et al., Antibodies, a Laboratory Manual(Cold Spring Harbor Labs Press, 1988), and include immunizing animals toproduce preparations of polyclonal antibodies that are recovered from,for example, ascites fluid and purified by methods known in the art toyield preparations that are reactive to IFNL4 protein. Many species haveproteins sharing related sequences and therefore it may be difficultusing standard immunization protocols to produce antibodies thatrecognize a protein from only one specie. Therefore, modification ofstandard methods used to produce antibodies, such as, for example,subtractive hybridization techniques, are also contemplated. Suchmodifications are known to those skilled in the art. In another method,antibodies for use in the present invention are produced recombinantlyusing techniques disclosed in Sambrook et al., Molecular Cloning: ALaboratory Manual, (Cold Spring Harbor Labs Press, 1989).

One embodiment of the present invention is an antibody that selectivelybinds to a variant of an IFNL4 protein. In one embodiment, the antibodyselectively binds a variant selected from the group consisting ofCys17Tyr), Arg60Pro) and Pro70Ser).

Other suitable methods include producing monoclonal antibodies. Briefly,monoclonal antibodies are produced from the fusion of spleen cells froman immunized animal and myeloma cells to produce a hybridoma. Hybridomascan be screened for production of the proper antibody, then cultured andthe antibodies harvested. Methods to produce and screen such hybridomasare known to those skilled in the art and are described in Harlow, etal., supra. Moreover, methods to prepare an antigen so that antibodiesproduced will be reactive with IFNL4 protein are known in the art andare described, for example, in Harlow, et al., supra. Preparation of theantigen material for injection into the animal includes any techniqueknown in the art, and include, for example, using the full-lengthprotein, using peptides selected from immunogenic regions of theprotein, modifying the antigen by methods such as, for example,dinitrophenol coupling, arsynyl coupling, denaturation of the antigen,coupling antigen to protein carriers such as, for example, keyholelimpet hemacyanin, peptides containing Class II-T-cell receptor bindingsites, to beads, and any other method known in the art. See Harlow, etal., supra.

Antibodies of the present invention can include multifunctionalantibodies, for example a bifunctional antibody having at least onefunctional portion that specifically binds to an IFNL4 protein. Suchmultifunctional antibodies can include, for example, a chimeric moleculecomprising a portion of the molecule that binds to an IFNL4 protein anda second portion that enables the chimeric molecule to be bound to asubstrate or to be detected in such a manner that the binding to theIFNL4 protein is unimpaired. Examples of suitable second portionsinclude, but are not limited to, a fragment of an immunoglobulinmolecule, a fluorescent protein or an enzyme.

An antibody used in the present invention can be contained in aformulation. For example, an antibody can be combined with a buffer inwhich the antibody is solubilized, and/or with a carrier. Suitablebuffers and carriers are known to those skilled in the art. Examples ofsuitable buffers include any buffer in which an antibody can function toselectively bind to an IFNL4 protein, such as, but not limited to,phosphate buffered saline, water, saline, phosphate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethansulfonic acid buffered saline) TESbuffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). Examples of carriers include, but are not limitedto, polymeric matrices, toxoids, and serum albumins, such as bovineserum albumin. Carriers can be combined with an antibody or conjugated(i.e. attached) to an antibody in such a manner as to not substantiallyinterfere with the ability of the antibody to selectively bind to anIFNL4 protein.

As has been described, IFNL4 protein is produced as a result of anindividual carrying a specific allele (i.e., the deletion allele) of thess469415590 polymorphism. As has also been described, the inventors havepreviously discovered that carrying at least one deletion allele of thess469415590 polymorphism increases the likelihood that an individualwill be unable to spontaneously clear an HCV infection, and thelikelihood that the individual will fail to respond to treatment of anHCV infection (as described in U.S. Provisional Application No.61/543,620, now International Application No. PCT/US12/59048, filed Oct.5, 2012). Thus, the presence, absence and/or level of IFNL4 proteinpresent in an individual can be used to determine the likelihood thatthe individual will spontaneously clear an HCV infection and/or thelikelihood the individual will respond to treatment for an HCVinfection.

Accordingly, one embodiment of the present invention is a method forpredicting the likelihood of an individual to spontaneously clear an HCVinfection by analyzing a biological sample from an individual todetermine the presence or absence of an IFNL4 protein of the presentinvention in the sample. The presence of an IFNL4 protein of the presentinvention in sample indicates that the individual is less likely tospontaneously clear an HCV infection.

In one embodiment, the absence of IFNL4 protein in the sample indicatesthe individual is predicted to be more likely to spontaneously clear anHCV infection. In one embodiment, the presence of IFNL4 protein in thesample indicates that the individual is predicted to be less likely tospontaneously clear an HCV infection.

Another embodiment of the present invention is a method for predictingthe likelihood that an individual will respond to a treatment for HCVinfection, by analyzing a biological sample from an individual todetermine the presence or absence of an IFNL4 protein of the presentinvention in the individual. The presence of an IFNL4 protein of thepresent invention indicates the likelihood that the individual will notrespond to treatment for an HCV infection, or benefit from theadministration of a treatment for an HCV infection.

In one embodiment, the absence of IFNL4 protein in the sample indicatesthe individual is predicted to be more likely to respond to treatmentfor an HCV infection. In one embodiment, the presence of IFNL4 proteinin the sample indicates that the individual is predicted to be lesslikely to respond to treatment for an HCV infection.

As has been described, the IFNL4 protein is encoded by a gene on humanchromosome 19. It will be appreciated by those skilled in the art thatbecause mammals have pairs of chromosomes, they have two copies of thisgene, the sequences of which are not necessarily identical. That is,while this region on one chromosome may contain one allele of apolymorphism (e.g., rs67272382), the same region of the other chromosomemay contain the same or a different allele of that polymorphism. Ininstances where two loci in an individual contain different sequences(e.g. an allele and the wild-type sequence, two different alleles), theindividual is referred to as being heterozygous for that loci. Ininstances where two loci in an individual contain the same sequence(e.g., both contain the same allele), the individual is referred to asbeing homozygous for that loci. The presence of one copy of apolymorphism can have a different affect on the likelihood ofspontaneously clearing an HCV infection, or responding to an HCVtreatment, than the presence of two copies of the same polymorphism. Forexample, if both chromosomes contain the insertion allele (and thereforelack the deletion allele) the individual will not produce the IFNL4protein. However, if one chromosome contains the insertion allele, andthe sister chromosome contains the deletion allele, the individual willproduce some amount of IFNL4 protein. Finally, if an individual has twocopies of the deletion allele, such an individual may produce more IFNL4protein than does an individual that is heterozygous for that allele.Consequently, an individual who has two copies of the insertion alleleis more likely to spontaneously clear an HCV infection, or respond to atreatment for HCV infection, than is an individual who only has one copyof such an allele. Likewise, an individual who has one copy of aninsertion allele and one copy of a deletion allele is more likely tospontaneously clear an HCV infection, or respond to treatment for HCV,than is an individual who has two copies of the deletion allele.

In one embodiment, an individual who is heterozygous for an insertionallele of the present invention is more likely to clear an HCVinfection, or respond to an HCV treatment, than is an individual whodoes not carry such an allele. In another embodiment, an individual whois homozygous for an insertion allele of the present invention is morelikely to clear an HCV infection, or respond to an HCV treatment, thanis an individual who is heterozygous for such an allele. In oneembodiment, an individual who is heterozygous for a deletion allele ofthe present invention is less likely to spontaneously clear an HCVinfection, or respond to treatment for an HCV infection, than is anindividual who does not carry any deletion alleles of the presentinvention. In another embodiment, an individual who is homozygous for adeletion allele of the present invention is even less likely tospontaneously clear an HCV infection, or respond to treatment for an HCVinfection, than is an individual who is heterozygous for a deletionallele of the present invention.

Accordingly, one embodiment of the present invention is a method forpredicting the likelihood of an individual to spontaneously clear an HCVinfection, by analyzing a biological sample from an individual todetermine the level of IFNL4 mRNA or protein present in the sample, ifany. The level of IFNL4 protein present in the sample is indicative ofthe likelihood the individual to spontaneously clear an HCV infection.

Another embodiment of the present invention is a method for predictingthe likelihood of an individual to respond to treatment for an HCVinfection by analyzing a biological sample from individual in order todetermine the level of IFNL4 mRNA or protein present in the sample, ifany. The level of IFNL4 protein present in the sample is indicative ofthe likelihood that the individual to respond to a treatment for an HCVinfection.

The terms individual, subject, and patient are well-recognized in theart, and are herein used interchangeably to refer to a mammal, includingdog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and,most preferably, a human. In some embodiments, the subject is in need ofa hepatitis C treatment. For example, in one embodiment the subject isinfected with HCV. However, in other embodiments, the subject is notinfected with HCV. In one embodiment, the subject is at risk forinfection with HCV. In one embodiment, the subject has been exposed toHCV. As used herein, the terms exposed, exposure, and the like, indicatethe subject has come in contact with bodily fluid from anotherindividual who is infected with HCV. Contact can occur through suchthings as, for example, a needle stick, sexual contact, or the birthingprocess.

The terms individual, subject, and patient by themselves do not denote aparticular age, sex, race, and the like. Thus, individuals of any age,whether male or female, are intended to be covered by the presentdisclosure. Likewise, the methods of the present invention can beapplied to any race, including, for example, Caucasian (white),African-American (black), Native American, Native Hawaiian, Hispanic,Latino, Asian, and European. In some embodiments of the presentinvention, such characteristics are significant. In such cases, thesignificant characteristic(s) (age, sex, race, etc.) will be indicated.

The term hepatitis C virus, or HCV, is used herein to define an RNAviral species of which pathogenic strains cause hepatitis C, also knownas non-A, non-B hepatitis. Based on genetic differences between HCVisolates, the hepatitis C virus species is classified into six majorgenotypes (1-6) with several subtypes within each genotype. Subtypes arefurther broken down into quasi species based on their genetic diversity.The preponderance and distribution of HCV genotypes varies globally. Forexample, in North America, genotype 1a predominates followed by 1b, 2a,2b, and 3a. In Europe, genotype 1b is predominant followed by 2a, 2b,2c, and 3a. Genotypes 4 and 5 are found almost exclusively in Africa.The viral genotype may be clinically important in determining potentialresponse to interferon-based therapy and the required duration of suchtherapy. Genotypes 1 and 4 are generally less responsive tointerferon-based treatment than are the other genotypes (2, 3, 5 and 6).It is to be noted that genotypes 5 and 6 are rare in the U.S.population.

As used herein, hepatitis C is an infectious disease affecting theliver, which is caused by the hepatitis C virus (HCV). The initialinfection with HCV may produce acute symptoms or the individual may beasymptomatic (without symptoms), but once established, chronic hepatitisC infection can progress to scarring of the liver (fibrosis), andadvanced scarring (cirrhosis) which is generally apparent after manyyears. In some cases, those with chronic hepatitis C will go on todevelop liver failure or other complications of chronic hepatitis C,including liver cancer.

According to the present invention, chronic hepatitis C refers to aninfection with HCV that persists for more than six months. Clinically,it is often asymptomatic and it is often discovered accidentally. Thenatural course of chronic hepatitis C varies considerably from oneperson to another. Although almost all people infected with HCV haveevidence of inflammation on liver biopsy, the rate of progression ofliver scarring (fibrosis) shows significant variability amongindividuals. Accurate estimates of the risk over time are difficult toestablish because of the limited time that tests for this virus havebeen available.

In some embodiments, the individual is co-infected with at least oneother organism such as, for example, hepatitis B virus, hepatitis Avirus, staphylococcus aureus, and/or the human immunodeficiency virus(HIV).

As used herein, spontaneous clearance refers to the ability of aninfected individual to clear HCV from their blood without the need foradministration of a therapeutic treatment designed to aid suchclearance. If an individual is capable of spontaneously clearing an HCVinfection, such clearance is typically observed during an acuteinfection. Authoritative clinical reviews have generally quotedclearance rates as low as 10-15%.

As used herein, the terms treat, treatment, and the like, refer totherapeutic treatment and prophylactic treatment, or preventativemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological condition or disease, or obtain beneficial ordesired clinical results. In this regard, treatment refers to theadministration of a therapeutic agent to slow down or prevent anundesired physiological condition or disease, or the symptoms associatedwith the above conditions or diseases. In one embodiment, the treatmentis one that helps a patient responsive to such treatment reduce thelevel of HCV RNA present in their body. By association, such a reductionreflects a reduction in the level of HCV present in the patient's body.In one embodiment, the treatment reduces the amount of HCV RNA in thepatient's body by at least 50%, at least 75%, at least 85%, at least95%, or at least 99% during the course of treatment. In one embodiment,treatment completely eliminates HCV RNA from the patient.

With regard to treatments for HCV, methods of the present invention canbe used to predict the response to any therapeutic agent useful fortreating HCV infections. Generally, treatment for an HCV infection is aninterferon based treatment. Thus, in one embodiment, treatment is aninterferon-based treatment. In various preferred embodiments, theinterferon-based treatment is selected from the group comprising IFN-α,IFN-λ, or any pegylated-interferon. In another embodiment, theinterferon-based treatment is combined with ribavirin. In variousembodiments, further combinations can include antiprotease drugs and/orother antiviral drugs. In one embodiment, the treatment comprises IFN,ribavirin and an HCV protease inhibitor.

While a treatment for an HCV infection can be administered to help apatient clear an HCV infection, not all patients are responsive to suchtreatment. That is, in some patients, while the treatment may cause somereduction in the level of HCV RNA, it does not result in a sustainedvirological response. A patient for whom treatment does not result in asustained virological response is referred to as a non-responder.Likewise, a patient for whom treatment results in a sustainedvirological response is referred to as a responder. A sustainedvirological response is defined as the lack of HCV RNA in the blood 24weeks after the cessation of treatment. In determining such a result,the HCV RNA level is typically measured at several times points duringthe course of treatment in order to measure the treatment response. Thelower the HCV RNA level is at these time points, the more likely it isthat a patient will achieve a sustained virological response.

As used herein, predicting a clinical response refers to knowing thelikelihood that a patient will spontaneously clear an HCV infectionprior to, or during, the acute phase of infection with HCV. It alsorefers to knowing the likelihood that a treatment for HCV infection willcause a sufficient reduction in the level of HCV RNA in a patient,before the treatment is administered to the patient. With regard to thepresent invention, predicting the clinical response may also be referredto as determining the susceptibility of a patient to response, ornon-response, to a treatment for HCV infection, or the susceptibility ofa patient to spontaneously clearing the virus.

As used herein the terms susceptible, susceptibility, and the like,refer to the likelihood, or probability, an individual willspontaneously clear an HCV infection, or will respond to treatment forsuch an infection. Such likelihood can also be referred to as apredisposition. In the context of the present invention, the likelihoodof spontaneously clearing an HCV infection and/or responding to atreatment need not be absolute. That is, for example, while the presenceof IFNL4 protein in the individual decreases the likelihood that theindividual will spontaneously clear an HCV infection, or respond totreatment, in a population of patients, all of whom carry thess469415590 deletion allele (and therefore produce mRNA or IFNL4protein), some percentage of such population may spontaneously clear anHCV infection or respond to treatment. This is likely due to acombination of other factors such as, for example, the genotype of thevirus, race, age, gender, and the genetic makeup of the individual atloci other than those in the IFNL4 gene. Thus, in one embodiment, theabsence of IFNL4 protein in a sample from an individual indicates theindividual is more likely to spontaneously clear an HCV infection thanis a patient that produces the IFNL4 mRNA and protein. In oneembodiment, the absence of IFNL4 protein in a sample from an individualindicates the individual is more likely to respond to treatment for anHCV infection than is a patient that produces the IFNL4 protein. Thus, apatient that does not produce the IFNL4 protein is more likely tobenefit from administration of a treatment than is a patient thatproduces IFNL4. In other embodiments, the presence of IFNL4 protein in asample from an individual indicates the individual is less likely tospontaneously clear an HCV infection than is a patient that does notproduce IFNL4. In yet another embodiment, the presence of IFNL4 mRNA andprotein in a sample from an individual indicates the individual is lesslikely to respond to a treatment for an HCV infection than is a patientthat does not produce IFNL4 mRNA and protein. Thus, a patient thatproduces IFNL4 protein is less likely to benefit from administration ofa treatment than is a patient not having the particular.

Thus, it will be appreciated by those skilled in the art that,likelihood, susceptibility, predisposition, and the like, are relativeterms. Methods and terminology for quantifying and reporting thelikelihood of a patient to respond to treatment, or spontaneously clearan HCV infection, are known to those skilled in the art. For example,one such method is a relative indication determined by comparing thenumber of individuals that produce the IFNL4 mRNA and protein and thatspontaneously clear an HCV infection, with the number of individualsthat do not produce the IFNL4 mRNA and protein and that alsospontaneously clear HCV infection. A similar comparison can be madebetween individuals that produce the IFNL4 protein and who respond totreatment for an HCV infection, and the number of individuals that donot produce the IFNL4 protein and who respond to treatment for an HCVinfection. Such a relative comparison can be illustrated using a foldincrease; for example, 1.5 fold (1.5×), 2×, 3×, 5×, etc. Such relativecomparison can also be illustrated using a percent increase. Forexample, if the number of patients that do not produce the IFNL4 proteinand that respond to treatment, or spontaneously clear HCV, is twice thenumber of patients that produce the IFNL4 protein and that respond totreatment, or spontaneously clear the virus, it could be said thatindividuals lacking the IFANAN protein are 100% more likely to respondto treatment or spontaneously clear HCV. Thus, in various embodiments,an individual that does not produce IFNL4 protein is at least 1.5×(fold), 2.0×, 2.5×, 3.0×, 4.0×, or 5.0×. Thus if an individual isdetermined to be 2× more likely to spontaneously clear an HCV infection,such a relative number means that in a population representative of theindividual, it would be expected that twice as many individuals wouldspontaneously clear an HCV infection as the number of individuals thatwould not spontaneously clear an HCV infection.

Relative comparisons can also be illustrated using an odds ratio, whichis a statistical method for relative comparisons that is used whenselection of study subjects is based on the clinical outcome ofinterest. In one embodiment, the likelihood of an individualspontaneously clearing an HCV infection, or responding to a treatmentfor HCV, has an odds ratio of at least about 1.2, at least about 1.4, atleast about 1.6, at least about 1.8, at least about 2.0, at least about2.2, at least about 2.4, at least about 2.6, at least about 2.8, atleast about 3.0, at least about 3.2, at least about 3.4, at least about2.6, at least about 3.8, at least about 4.0, at least about 4.2, atleast about 4.4, at least about 4.6, at least about 4.8 or at leastabout 5.0. Methods of calculating an odds ratio are known to thoseskilled in the art and are exemplified in Rothman, Kenneth J.;Greenland, Sander; Lash, Timothy L. (2008). Modern Epidemiology,Lippincott Williams & Wilkins, Third edition, which is incorporatedherein in its entirety.

In various embodiments, the level of IFNL4 protein in the sample iscompared to reference levels of IFNL4 protein (also referred to asreference standards). Such reference standards can be obtained fromvarious sources. For example, the reference standard can be from anindividual known to be able to spontaneously clear an HCV infection orrespond to treatment for an HCV infection. In another example, thereference standard can be from an individual known to be unable tospontaneously clear an HCV infection or respond to treatment for an HCVinfection. Ideally, reference standards represent the average level ofprotein present in a population of individuals that can, or cannot,clear an HCV infection or respond to treatment for an HCV infection.

In one embodiment, if the level of IFNL4 protein present in the samplefrom a first individual is less than the level of IFNL4 protein presentin a reference standard from a second individual, known to be able toclear an HCV infection or respond to treatment for an HCV infection, ora population of such individuals, then the first individual is predictedto be able to clear an HCV infection or respond to treatment for an HCVinfection. In one embodiment, if the level of IFNL4 protein present inthe sample from a first individual is greater than the level of IFNL4protein present in a reference standard from a second individual, knownto be able to clear an HCV infection or respond to treatment for an HCVinfection, or a population of such individuals, then the firstindividual is predicted to be unable to clear an HCV infection orrespond to treatment for an HCV infection.

As used herein, a biological sample refers to any fluid or tissue froman individual that can be analyzed the activity of the IFNL4 protein,including expression level, protein level or a biological activity ofthe IFNL4 protein. Examples of the type of sample that can be used topractice the present invention include, but are not limited to, a bloodsample, a urine samples, a tear sample, a tissue sample, and a buccalswab. Preferred samples for extracting DNA and genotyping are blood andbuccal swab samples. Samples useful for detecting the presence, absenceor level of mRNA and protein levels are known to those skilled in theart. Moreover, methods of obtaining such samples are also known to thoseskilled in the art. IFNL4 expression may be determined in liver biopsiesof carriers of a risk genotype and infected by HCV.

Once a sample has been obtained, it is analyzed to determine thepresence, absence or level of IFNL4 mRNA and proteins of the presentinvention. As used herein, the terms “determine,” “determine the levelof IFNL4 mRNA and protein,” “determine the amount of IFNL4 mRNA andprotein,” “determine the IFNL4 mRNA and protein level”, and the like,are meant to encompass any technique which can be used to detect ormeasure the presence of IFNL4 in a sample. In this context, IFNL4 is anexample of an analyte. Such techniques may give qualitative orquantitative results. IFNL4 levels can be determined by detecting theentire IFNL4 mRNA and protein or by detecting fragments, degradationproducts or reaction products of IFNL4. In a preferred method, the levelof IFNL4 is determined using a suitable IFNL4-binding compound.

In the case of a method for determining the likelihood of a response toa treatment for an HCV infection, or resistance to such treatment,according to the present invention, the presence, absence or level of anIFNL4 protein of the present invention indicates the likelihood of theindividual to respond to such treatment. In the case of a method ofdetermining the likelihood of spontaneously clearing an HCV infection,according to the present invention, the presence, absence or level of anIFNL4 protein of the present invention indicates the likelihood of theindividual spontaneously clearing an HCV infection.

Any known method of analyzing a sample for an analyte can be used topractice the present invention, so long as the method detects thepresence, absence, or amount of IFNL4 protein. Examples of such methodsinclude, but are not limited to, immunological detection assays andnon-immunological methods (e.g., enzymatic detection assays). In animmunological detection assay, the sample to be tested for the presence,absence or level of an analyte is contacted with a binding molecule,such as an antibody. As used herein, the term contact, contacted,contacting, and the like, refers to the introduction of a sampleputatively containing IFNL4 to an IFNL4-binding compound, for example,by combining or mixing the sample with the IFNL4-binding compound. Oneexample of an IFNL4-binding compound is an antibody that selectivelybinds to IFNL4. However, other molecules that bind to IFNL4 are alsoencompassed. For example, a receptor to which IFNL4 binds can be used asan IFNL4-binding compound in assays of the present invention.

When IFNL4 is present in the sample, an IFNL4-compound complex is thenformed. Such complex formation refers to the ability of an IFNL4-bindingcompound to selectively bind to the IFNL4 in order to form a stablecomplex that can be detected. Detection can be qualitative,quantitative, or semi-quantitative. Binding of IFNL4 in the sample tothe IFNL4-binding compound is accomplished under conditions suitable toform a complex. Such conditions (e.g., appropriate concentrations,buffers, temperatures, reaction times) as well as methods to optimizesuch conditions are known to those skilled in the art. Binding can bemeasured using a variety of methods standard in the art including, butnot limited to, enzyme immunoassays (e.g., ELISA), immunoprecipitations,immunoblot assays and other immunoassays as described, for example, inSambrook et al., supra, and Harlow, et al., supra. These references alsoprovide examples of complex formation conditions.

In one embodiment, the IFNL4/IFNL4-binding compound complex, alsoreferred to herein as an IFNL4-compound complex, or simply the complex,can be formed in solution. In another embodiment, a complex can beformed while the IFNL4-binding compound is immobilized on (e.g., coatedonto) a substrate. Immobilization techniques are known to those skilledin the art. Suitable substrate materials include, but are not limitedto, plastic, glass, gel, celluloid, fabric, paper, and particulatematerials. Examples of substrate materials include, but are not limitedto, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF(polyvinylidene-fluoride), and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a microtiter plate, a dipstick, a strip, a bead,a lateral flow apparatus, a membrane, a filter, a tube, a dish, acelluloid-type matrix, a magnetic particle, and other particulates.Particularly preferred substrates include, for example, an ELISA plate,a dipstick, an immunodot strip, a radioimmunoassay plate, an agarosebead, a plastic bead, a latex bead, a sponge, a cotton thread, a plasticchip, an immunoblot membrane, an immunoblot paper and a flow-throughmembrane. In one embodiment, a substrate, such as a particulate, caninclude a detectable marker. For descriptions of examples of substratematerials, see, for example, Kemeny, D. M. (1991) A Practical Guide toELISA, Pergamon Press, Elmsford, N.Y. pp 33-44, and Price, C. andNewman, D. eds. Principles and Practice of Immunoassay, 2nd edition(1997) Stockton Press, NY, N.Y., both of which are incorporated hereinby reference in their entirety.

In one embodiment, an IFNL4-binding compound is immobilized on asubstrate, such as a microtiter dish well, a dipstick, an immunodotstrip, or a lateral flow apparatus. A sample collected from anindividual is applied to the substrate and incubated under conditionssuitable (i.e., sufficient) to allow the formation of a complex betweenthe binding compound and any IFNL4 present in the sample.

In accordance with the present invention, once formed, the complex isthen detected. As used herein, the term “detecting complex formation”refers to identifying the presence of IFNL4-binding compound complexedto IFNL4. If complexes are formed, the amount of complexes formed can,but need not be, quantified. Complex formation, or selective binding,between a putative IFNL4-composition with an IFNL4-binding compound canbe measured (i.e., detected, determined) using a variety of methodsstandard in the art (see, for example, Sambrook et al. supra.), examplesof which are disclosed herein. A complex can be detected in a variety ofways including, but not limited to use of one or more of the followingassays: an enzyme-linked immunoassay, a competitive enzyme-linkedimmunoassay, a radioimmunoassay, a fluorescence immunoassay, achemiluminescent assay, a lateral flow assay, a flow-through assay, anagglutination assay, a particulate-based assay (e.g., using particulatessuch as, but not limited to, magnetic particles or plastic polymers,such as latex or polystyrene beads), an immunoprecipitation assay, aBIACORE™ assay (e.g., using colloidal gold), an immunodot assay (e.g.,CMG's Immunodot System, Fribourg, Switzerland), and an immunoblot assay(e.g., a western blot), an phosphorescence assay, a flow-through assay,a chromatography assay, a PAGE-based assay, a surface plasmon resonanceassay, a spectrophotometric assay, a particulate-based assay, and anelectronic sensory assay. Such assays are well known to those skilled inthe art.

Assays can be used to give qualitative or quantitative results dependingon how they are used. The assay results can be based on detecting theentire IFNL4 molecule or fragments, degradation products or reactionproducts of IFNL4. Some assays, such as agglutination, particulateseparation, and immunoprecipitation, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker.

In other assays, conjugation (i.e., attachment) of a detectable markerto the anti-IFNL4 compound or to a reagent that selectively binds to theanti-IFNL4 compound aids in detecting complex formation. A detectablemarker can be conjugated to the anti-IFNL4 compound or reagent at a sitethat does not interfere with ability of the anti-IFNL4 compound to bindIFNL4 protein. Methods of conjugation are known to those of skill in theart. Examples of detectable markers include, but are not limited to, aradioactive label, a fluorescent label, a chemiluminescent label, achromophoric label, an enzyme label, a phosphorescent label, anelectronic label; a metal sol label, a colored bead, a physical label,or a ligand. A ligand refers to a molecule that binds selectively toanother molecule. Preferred detectable markers include, but are notlimited to, fluorescein, a radioisotope, a phosphatase (e.g., alkalinephosphatase), biotin, avidin, a peroxidase (e.g., horseradishperoxidase), beta-galactosidase, and biotin-related compounds oravidin-related compounds (e.g., streptavidin or IMMUNOPURE™NeutrAvidin).

Means of detecting such markers are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters; fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic markers aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric markers are detected by simplyvisualizing the colored label.

In one embodiment, an IFNL4-compound complex can be detected bycontacting a sample with an antibody specific for the compound, whereinthe antibody is conjugated to a detectable marker. A detectable markercan also be conjugated to an anti-IFNL4 antibody or other compound whichbinds the IFNL4-binding-compound in such a manner as not to block theability of the anti-compound antibody or other compound to bind to theIFNL4-binding compound being detected. Preferred detectable markersinclude, but are not limited to, fluorescein, a radioisotope, aphosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase(e.g., horseradish peroxidase), beta-galactosidase, and biotin-relatedcompounds or avidin-related compounds (e.g., streptavidin or IMMUNOPURE™NeutrAvidin).

In another embodiment, a complex is detected by contacting the complexwith an indicator molecule. Suitable indicator molecules includemolecules that can bind to the IFNL4/IFNL4-binding molecule complex orto the IFNL4 protein. As such, an indicator molecule can comprise, forexample, an IFNL4-binding reagent, such as an antibody. Preferredindicator molecules that are antibodies include, for example, antibodiesreactive with the antibodies from species of animal in which theanti-IFNL4 antibodies are produced. An indicator molecule itself can beattached to a detectable marker of the present invention. For example,an antibody can be conjugated to biotin, horseradish peroxidase,alkaline phosphatase or fluorescein.

The present invention can further comprise one or more layers and/ortypes of secondary molecules or other binding molecules capable ofdetecting the presence of an indicator molecule. For example, anuntagged (i.e., not conjugated to a detectable marker) secondaryantibody that selectively binds to an indicator molecule can be bound toa tagged (i.e., conjugated to a detectable marker) tertiary antibodythat selectively binds to the secondary antibody. Suitable secondaryantibodies, tertiary antibodies and other secondary or tertiarymolecules can be readily selected by those skilled in the art. Preferredtertiary molecules can also be selected by those skilled in the artbased upon the characteristics of the secondary molecule. The samestrategy can be applied for subsequent layers.

Preferably, the indicator molecule is conjugated to a detectable marker.A developing agent is added, if required, and the substrate is submittedto a detection device for analysis. In some protocols, washing steps areadded after one or both complex formation steps in order to removeexcess reagents. If such steps are used, they involve conditions knownto those skilled in the art such that excess reagents are removed butthe complex is retained.

One embodiment of the present invention involves the use of a lateralflow assay, examples of which are described in U.S. Pat. No. 5,424,193,issued Jun. 13, 1995, to Pronovost et al.; U.S. Pat. No. 5,415,994,issued May 16, 1995, by Imrich et al; WO 94/29696, published Dec. 22,1994, by Miller et al.; and WO 94/01775, published Jan. 20, 1994, byPawlak et al.; all of which are incorporated by reference herein. Alateral flow assay is an example of a single-step assay. In asingle-step assay, once the sample has been obtained and made ready fortesting, only a single action is necessary on the part of the user todetect the present of an analyte. For example, the sample, in whole orpart, can be applied to a device that measures analyte in the sample. Inone embodiment, a sample is placed in a lateral flow apparatus thatincludes the following components: (a) a support structure defining aflow path; (b) a labeling reagent comprising a bead conjugated to aspecific antibody, the labeling reagent being impregnated within thesupport structure in a labeling zone; and (c) a capture reagent.Preferred antibodies include those disclosed herein. The capture reagentis located downstream of the labeling reagent within a capture zonefluidly connected to the labeling zone in such a manner that thelabeling reagent can flow from the labeling zone into the capture zone.The support structure comprises a material that does not impede the flowof the beads from the labeling zone to the capture zone. Suitablematerials for use as a support structure include ionic (i.e., anionic orcationic) material. Examples of such a material include, but are notlimited to, nitrocellulose, PVDF, or carboxymethylcellulose. The supportstructure defines a flow path that is lateral and is divided into zones,namely a labeling zone and a capture zone. The apparatus can furtherinclude a sample receiving zone located along the flow path, preferablyupstream of the labeling reagent. The flow path in the support structureis created by contacting a portion of the support structure downstreamof the capture zone, preferably at the end of the flow path, to anabsorbent capable of absorbing excess liquid from the labeling andcapture zones.

In another embodiment, a lateral flow apparatus used to detect IFNL4includes: (a) a support structure defining a flow path; (b) a labelingreagent comprising a anti-IFNL4 antibody as described above, thelabeling reagent impregnated within the support structure in a labelingzone; and (c) a capture reagent, the capture reagent being locateddownstream of the labeling reagent within a capture zone fluidlyconnected to the labeling zone in such a manner that the labelingreagent can flow from the labeling zone into the capture zone. Theapparatus preferably also includes a sample receiving zone located alongthe flow path, preferably upstream of the labeling reagent. Theapparatus preferably also includes an absorbent located at the end ofthe flow path. One preferred embodiment includes a capture reagentcomprising anti-IFNL4 antibody.

One embodiment of the present invention is a “dipstick” device that candetect IFNL4 in individuals. Dipsticks may be constructed in a varietyof ways that partly depend on the way in which they will be used. Theymay be held directly in a sample (e.g., a urine stream), dipped directlyin sample contained in a collection vessel, or have sample applied to astrip contained in a plastic cassette or platform. Another example of adipstick is a “flow-through” device, an example of which is aheterogenous immunometric assay system based on a capture antibodyimmobilized onto a membrane attached to an absorbent reservoir, A “bead”refers to a particulate substrate composed of a matrix such as latex orpolystyrene, which can be covalently or non-covalently cross-linked to adetection molecule. A preferred embodiment of the “dipstick” assay is animmunometric system, described in U.S. Pat. No. 5,656,502, issued onAug. 12, 1997, to MacKay and Fredrickson, and U.S. Pat. No. 6,001,658,issued Dec. 14, 1999 to Fredrickson, both incorporated herein byreference. Particularly preferred is an IMMUNODIP™ device available fromDiagnostic Chemicals Ltd., PEI, CA.

Once a sample has been analyzed to determine the presence, absence orlevel of IFNL4, the individual can be selected, or identified, as beingable to, or not being able to, spontaneously clear an HCV infection orrespond to treatment for such an infection. Such a selection is madeusing the results from the analysis step of the disclosed method. Forexample, a person obtaining the result of the analysis step could thendecide if the person is able to respond to a treatment for HCV infectionand, if not, decide to use an alternative treatment. As a furtherexample, a person reviewing the data obtained from the analysis stepcould then decide if the person is able to spontaneously clear an HCVinfection and, if not, decide to begin administration of a treatment. Inone embodiment, the selection is made using a device. For example, adevice could be designed such that when the IFNL4 protein is present inthe sample, the output of the device indicates the individual is unableto spontaneously clear an HCV infection or respond to treatment for suchan infection. In one embodiment, the device is an electronic device. Forexample, a device that analyzes the results of an ELISA assay could bedesigned to display the result with regard to the ability of anindividual to spontaneously clear an HCV infection or respond totreatment for such an infection. In one embodiment, the device comprisesa microprocessor. In one embodiment, the device is a computer.

It has been disclosed herein that when IFNL4 is created by thess469415590-AG allele, the protein may carry non-synonymous variantsthat affect the biological activity of the protein. Examples of suchvariants include (rs73555604 (Cys17Tyr) in exon 1, rs142981501(Arg60Pro) and rs117648444 (Pro70Ser) in exon 2). It will be appreciatedby those skilled in the art that such variations in sequence, whenpresent in the coding portion of the IFNL4 locus, may affect IFNL4activity and thereby alter the overall impact of an individuals abilityto clear the virus or respond to treatment. For example, in a limitedpopulation study, the presence of the rs117648444 (Pro70Ser) appeared tocorrelate with an increased rate of spontaneous clearance as compared toindividuals who lacked this variant. Further, this variant has asignificantly different Heatmap profile (FIG. 21) as compared to theprofiles of rs142981501 (Arg60Pro) and rs117648444 (Pro70Ser). Thus, inone embodiment, if IFNL4 is detected, the method comprises the furtherstep of analyzing the detected IFNL4 for the present of one or moresequence variations, wherein the presence of one or more sequencevariations is indicative of the likelihood that an individual willspontaneously clear an HCV infection and/or the likelihood an individualwill respond to treatment for an HCV infection.

Since methods of the present invention can be used to predict anindividual's response to a therapeutic treatment, such methods can beincorporated into a treatment plan. Thus, one embodiment of the presentinvention is a method for treating a patient suffering from a chronichepatitis C virus infection, by analyzing a biological sample from anindividual to determine the presence, absence or level of IFNL4 proteinpresent in the biological sample. The decision to administer treatmentfor a hepatitis C virus infection to the individual is based on thepresence, absence or amount of IFNL4 protein present in the sample.

In one embodiment, the patient has an acute HCV infection. In oneembodiment, the patient has a chronic HCV infection. In one embodiment,the absence of IFNL4 protein in the sample indicates the individual islikely to respond to a treatment for an HCV infection and thus atreatment is administered. In another embodiment, the presence of IFNL4protein in the sample indicates that the individual is unlikely torespond to administration of a treatment for an HCV infection, andtreatment is not administered. In one embodiment, a level of IFNL4protein present in the sample is less than the level observed in areference sample from a second individual, or pool of individuals, knownto respond to treatment, indicating that the individual is likely torespond to a treatment for an HCV infection and thus a treatment isadministered. In one embodiment, a level of IFNL4 protein present in thesample is higher than the level observed in a reference sample from asecond individual, or pool of individuals, known to be unable to respondto treatment or otherwise lacking in response to a treatment for an HCVinfection, indicating that the individual is unlikely to respond to atreatment for an HCV infection, and thus a treatment is administered.

The determination of IFNL4 levels in a patient suffering from chronichepatitis C will enable medical personnel to establish the besthepatitis C treatment regimen for that patient (e.g., nature, dose andduration of hepatitis C treatment and/or other antiviral drugs). Forexample, if the above method reveals that the patient produces IFNL4protein, indicating that said subject is unlikely to respond to ahepatitis C treatment, then this subject can be considered as goodcandidate for newer treatment strategies (such as therapy with higherdoses of currently available drugs, longer treatment duration withcurrently available drugs and/or newer drugs).

The present invention also covers additional methods of treatment usingmolecules of the present invention. For example, as will be appreciatedby now, the presence of IFNL4 protein in an individual results in theindividual being unable to spontaneously clear an HCV infection, orrespond to treatment for such an infection. Without being bound bytheory, the inventors believe that such an effect results from actionsof the IFNL4 protein in one or more immunological pathways, for example,activation of the JAK/STAT pathway. Consequently, elimination of suchactions should remove the effect. Thus, individuals producing IFNL4, andthus unable to spontaneously clear an HCV infection, or respond totreatment for such an infection, upon administration of a therapeuticcompound that eliminates IFNL4 activity should be able to spontaneouslyclear an HCV infection, or respond to treatment for such an infection.Accordingly, one embodiment of the present invention is a method totreat a patient for an HCV infection, the method comprisingadministering a compound to the patient, wherein the compoundselectively binds to IFNL4 protein in the patient, and wherein suchbinding results in reducing or eliminating IFNL4 activity in thepatient.

One example of such a therapeutic compound is an antibody of the presentinvention that selectively binds to IFNL4. Administration of such anantibody will result in the antibody binding and forming a complex withIFNL4. Such binding would eliminate IFNL4 activity in at least one oftwo ways. Physical binding of the antibody to IFNL4 can result in actualphysical inhibition of IFNL4 activity. Second, IFNL4-antibody complexeswill be recognized by the body and removed there from. IFNL4-bindingcompounds identified using methods of the present invention can also beused to eliminate or reduce IFNL4 activity in a patient.

In addition to IFNL4-binding compounds, reduction of IFNL4 activity canbe achieved by reducing or eliminating the production of mRNAtranscripts that encode IFNL4. Such reduction or elimination can beachieved by administration of small interfering RNAs that are specificfor mRNA molecules that encode IFNL4. Accordingly, one embodiment of thepresent invention is a method to treat a patient for an HCV infection,the method comprising administering a small interfering RNA to thepatient, wherein the small interfering RNA that is selective for mRNAencoding IFNL4, whereby such administration results in reducing oreliminating IFNL4 activity in the patient.

The present invention also encompasses methods of identifying compoundsthat regulate IFNL4 activity. Such compounds are referred to asregulators. For example, as previously discussed, the inventors haveshown that IFNL4 activates the JAK/STAT pathway, a reaction that caneasily be performed using an ELISA assay format. Thus, regulators ofIFNL4 activity can be identified by performing a JAK/STAT pathwayactivation assay using IFNL4 proteins of the present invention, andadding to the assay a test compound. The results of such assay are thencompared to the results of a second JAK/STAT activation assay that lacksany test compound, or to which has been added a compound that is knownhave no effect on IFNL4 activity. Accordingly, one embodiment of thepresent invention is a method for identifying regulators of IFNL4protein activity, by incubating IFNL4 protein under conditions thatallow measurement of IFNL4 protein activity and measuring the resultantIFNL4 protein activity. The IFNL4 protein can then be incubated in thepresence of a test compound under the same conditions used in theinitial protein activity assay and the resulting IFNL4 protein activityis measured. The two protein activities are then compared. If thedifference between the IFNL4 activity obtained in the absence and thepresence of the test compound is statistically significant, the testcompound is identified as a regulator of IFNL4 activity.

As used herein, regulators refer to compounds that affect the functionalactivity of an IFNL4 protein in vitro and/or in vivo. Regulators can beagonists and antagonists of an IFNL4 protein and can be compounds thatexert their effect on the IFNL4 protein via the expression, viapost-translational modifications, by direct interaction with the IFNL4protein or by other means. Agonists of IFNL4 protein are molecules that,when bound to IFNL4 protein increase or prolong the functional activityof the protein. Agonists of IFNL4 protein include proteins, nucleicacids, carbohydrates, small molecules, or any other molecule thatactivate IFNL4 protein. Antagonists of IFNL4 protein are molecules that,when bound to IFNL4 protein, decrease the amount or the duration of thefunctional activity of the protein. Antagonists include proteins,nucleic acids, carbohydrates, antibodies, small molecules, or any othermolecule that decrease the activity of IFNL4 protein.

The term modulate, as it appears herein, refers to a change in theactivity of IFNL4 protein. For example, modulation may cause an increaseor a decrease in functional activity, binding characteristics, or anyother biological, functional, or immunological properties of IFNL4protein.

Regulators of IFNL4 activity are identified either in assays that employcells that expressing IFNL4, either naturally or as a result of geneticmanipulation (e.g., recombinant cells) (cell-based assays) or in assayswith isolated IFNL4 protein (cell-free assays). The various assays canemploy a variety of variants of IFNL4 protein (e.g., full-length IFNL4protein, a biologically active fragment of IFNL4 protein, or a fusionprotein that includes all or a portion of IFNL4 protein). The assay canbe a binding assay entailing direct or indirect measurement of thebinding of a test compound or a known ligand for IFNL4 protein. Theassay can also be an activity assay entailing direct or indirectmeasurement of the IFNL4 protein activity. The assay can also be anexpression assay entailing direct or indirect measurement of theexpression of mRNA encoding IFNL4 protein, or the IFNL4 protein itself.

Suitable test compounds for use in the screening assays of the inventioncan be obtained from any suitable source, e.g., conventional compoundlibraries. The test compounds can also be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. Examples of methods for the synthesisof molecular libraries can be found in the art. Libraries of compoundsmay be presented in solution or on beads, bacteria, spores, plasmids orphage.

Also included in the present invention are kits useful for practicingthe disclosed methods of the present invention. Thus, one embodiment ofthe present invention is a kit for determining the likelihood ofresponse to a hepatitis C treatment in a subject, in accordance with thepresent invention, said kit comprising i) reagents for selectivelydetecting the presence, absence or level of, at least, IFNL4 mRNA orprotein in a sample obtained from the subject and ii) instructions forusing the kit.

One embodiment of the present invention is a kit for determining thelikelihood of spontaneous clearance of hepatitis C virus in a subjectinfected with the virus, in accordance with the present invention, saidkit comprising i) reagents for selectively detecting the presence,absence or level of, at least, IFNL4 protein in a sample obtained fromthe subject and ii) instructions for using the kit.

Kits of the present invention will contain at least some of the reagentsrequired to determine the presence, absence or level of IFNL4 protein.Reagents for kits of the present invention can include, but are notlimited to, isolated nucleic acid molecules of ht present invention, anIFNL4 protein of the present invention, and an IFNL4-binding compound(e.g., an antibody that selectively binds to IFNL4). In someembodiments, the IFNL4 protein and/or IFNL4-binding compounds may befixed to a solid substrate. The kits may further comprise controlproteins. One skilled in the art will, without undue experiments, beable to select the necessary reagents from the disclosure herein, inaccordance with the usual requirements. Reagents of the kit may alsocomprise a molecular label or tag.

Kits of the present invention can also comprise various reagents, suchas buffers, necessary to practice the methods of the invention, as knownin the art. These reagents or buffers may, for example, be useful toextract and/or purify the IFNL4 mRNA/protein from the biological sampleobtained from the subject. The kit may also comprise all the necessarymaterial such as microcentrifuge tubes necessary to practice the methodsof the invention.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

It should be appreciated that although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The publications and applications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

EXAMPLES Example 1. Analysis of the Inducible Expression Landscape inthe IFNL3 (IL28B) Region in Primary Human Hepatocytes Activation ofNormal Human Hepatocytes

Primary cultures of freshly isolated, normal human hepatocytes fromHCV-non-infected livers were purchased from Lonza Inc. (Walkersville,Md.). The cells were plated onto six-well, collagen-coated, tissueculture plates and showed high viability. The cells were then treatedwith PolyI:C at a final concentration of 50 μg/mL for 0, 1, 2, 4, 8 or24 hours. PolyI:C (polyinosinic:polycytidylic acid) is a Toll-likereceptor-3 (TLR3) agonist that simulates viral infection (Alexopoulou L,Holt A C, Medzhitov R, Flavell R A. (2001) Recognition ofdouble-stranded RNA and activation of NF-kappa B by Toll-like receptor3. Nature 413(6857):732-8). PolyI:C is structurally similar todouble-stranded RNA, which represents an intermediate phase in thereplication cycle of many types of viruses. PolyI:C has been usedextensively as a synthetic agent to mimic viral infection, and itinduces expression of both type-I (IFN-α) and type-III (IFN-λ)interferons in many cell types (Doyle S E, Vaidya S A, O'Connell R,Dadgostar H, Dempsey P W, Wu T, Rao G, Sun R, Haberland M E, Modlin R L,Cheng G. (2002) IRF3 mediates a TLR3/TLR4-specific antiviral geneprogram. Immunity 17(3):251-63; Doyle S E, O'Connell R, Vaidya S A, ChowE K, Yee K, Cheng G. (2003) Toll-like receptor 3 mediates a more potentanti-viral response than Toll-like receptor 4. J. Immunol.170(7):3565-71). After treating the hepatocyte cultures with PolyI:C forthe indicated time periods, the cells were harvested, and DNA and RNAextracts were prepared by standard methods. The quality and quantity ofDNA and RNA was measured by Nanodrop and Bioanalyzer analysis.

RNA-Sequencing

One microgram of total RNA was used for each of the hepatocyte samplestreated with PolyI:C for 0, 1, 2, 4, 8, 24 hours. After PolyA mRNAselection, the RNA samples were fragmented and ligated to 65 bp adaptorsto prepare paired end (PE) cDNA libraries with fragments of 200-250 bp,according to a standard Illumina protocol. The libraries were enrichedby 12 PCR cycles and sequenced at a concentration of 4.5 pM, using oneGenome Analyzer (GAII) lane per sample, generating 107 bp sequencingreads. In average, 47.2±6.6 million PE RNA-seq reads were generated persample. The human reference genome for the analysis was built based onUCSC hg19 index using Bowtie software. The sequenced reads wereprocessed using Illumina Pipeline OLB 1.9.0 and CASAVA 1.7.0 and alignedto the reference genome using TopHat v1.2.0 after removing some readsaccording to standard quality control settings. A library of all humantranscripts from Ensemble database, version GRCh37.61:useast.ensembl.org/info/data/ftp/index, was used to generate a libraryof exon junctions and reconstruct splicing forms. Default TopHatalgorithm removes from analysis RNA-seq reads that map to more than onegenomic region. Considering complexity of the region surrounding IFNL3(IL28B) gene, we implemented a special strategy. To allow non-exclusivemapping of reads to regions of high similarity, such as IFNL1 (IL29),IFNL2 (IL28A), IFNL3 (IL28B), we changed TopHat settings to allowmultiple alignments (up to 10). The mapping identified expressionclusters, representing potential exons. Based on the created database ofpotential splice junctions, previously unmapped reads were re-mapped byTopHat v1.2.0. To detect novel transcripts, the final aligned read fileswere processed by Cufflinks v0.9.3. Relative abundances of transcriptswere measured with fragments per kilobase of exon per million fragmentsmapped algorithm (FPKM). Confidence intervals for FPKM estimates werecalculated using a Bayesian inference method. In the presence ofsplicing forms, the highest expressing form of each gene was assigned aratio 1, and all other forms expressed at least at 1% level of the mainform (ratio >0.01) were used for analysis. The TopHat alignmentalgorithm brakes sequence reads into 25-bp segments that areindependently mapped and reconstructed back into sequence reads if allindividual segments are mapped correctly. To discover possible geneticvariants, we allowed 2 or 3 mismatches per segment. Mapping results werevisualized using both the UCSC genome browser and a local copy of theIntegrative Genomics Viewer (IGV) software: broadinstitute.org/igv/.Genetic variants in the region were visualized by IGV and then examinedmanually. The results of this analysis are shown in FIG. 1.

Detailed analysis of the 150 Kb region surrounding the IFNL3 (IL28B)gene showed no expression of known IFN-λ, genes IFNL1 (IL29), IFNL2(IL28A), IFNL3 (IL28B) after 0 or 1 hour of treatment, but strongactivation of these genes after 2-24 hours of treatment (FIG. 1, 2).This analysis also resulted in the identification of a novel, ˜2.5 kblong transcribed region located near rs12979860 (FIG. 1, 2, 3).Expression of this region was not detected without, or after 1 hour of,PolyI:C treatment, but was induced after 2, 4 or 8 hours and thenstrongly decreased at 24 hours. Analysis of paired-end RNA-seq readsidentified one splice junction site present in all of the putativetranscripts in this region. This sequence was used as a starting pointfor 5′RACE analysis. Such analysis resulted in the identification of asingle transcription start site, followed by a protein translation startsite 277 bp downstream (FIG. 3).

Example 2. Analysis of IFNL4 mRNA Expression

An allele-specific TaqMan assay for quantification of IFNL4 mRNA wasdesigned with the Primer Express Software and manufactured by AppliedBiosystems on demand. The assay consists of primers:

(SEQ ID NO: 13) IFNL4-p179_forw: GCCTGCTGCAGAAGCAGAGAT; (SEQ ID NO: 14)IFNL4-p179_rev: AGCCGAGCGCAGGACGA;and probes

(SEQ ID NO: 15) IFNL4_VIC AA (non-risk allele): ATCGCAGAAGGCC and(SEQ ID NO: 16) IFNL4 FAM-C (risk allele): ATCGCAGCGGCCC.

This assay should provide a fragment of 255 bp with cDNA and no productshould be obtained using DNA. The assay is highly specific for thetranscript encoding IFNL4-p179 and doesn't detect any other transcripts(IFNL4-p131, p107, etc). FAM fluorofore labels the deletion allele andthus is specific for IFNL4-p179 expression, while detection with VICfluorofore serves as a negative control, as no IFNL4 p179 should not bedetected in mRNA transcripts with the insertion allele.

Due to very high specificity, this assay allows detection even innon-DNAse I treated RNA samples. An alternative expression assay is:

(SEQ ID NO: 17) IFNL4_alt_F: GCCTGCTGCAGAAGCAGAGAT; (SEQ ID NO: 18)IFNL4_alt_R: GCTCCAGCGAGCGGTAGTG;and probes

(SEQ ID NO: 19) IFNL4_alt_VIC AA (non-risk allele): ATCGCAGAAGGCC;(SEQ ID NO: 20) IFNL4_alt_FAM-C (risk allele): ATCGCAGCGGCCC.This assay is has higher efficiency but is less specific thanIFNL4-p179. FAM fluorofore detects expression of all forms with riskallele and VIC—all forms with non-risk allele (VIC).

Example 3. Identification and Sequence Analysis of Novel IFNL4 Gene

The full-length open reading frame for IFNL4 transcript was PCRamplified from cDNA of PolyI:C-treated primary human hepatocytes withprimers:

(SEQ ID NO: 21) IFNL4 forw: ATGCGGCCGAGTGTCTGGGCC; (SEQ ID NO: 22)IFNL4 rev: GAGGCAAGGCCCAGAGTGTGCAG.

Additional sequences were added to primers for cloning into specificvectors. Due to very high GC content of this amplicon, PCR reactionswere performed with AmpliTaq Gold 360 Master Mix and 360 GC Enhancerusing the touchdown PCR program which includes initial denaturation stepwith 10 minutes at 95° C., followed by 20 cycles (30 seconds at 95° C.,45 seconds for 2 cycles at each temperature from 70° through 60° C.decreasing by 1° C. at each step, 45 seconds at 72° C.); 25 additionalcycles (30 seconds at 95° C., 45 seconds at 60° C. and 45 seconds at 72°C.); and final extension time of 7 minutes at 72° C.

Gel-purified PCR fragments (FIG. 5) were cloned into a C-terminalpFC14A-Halo tag expression vector (Promega) and sequenced forvalidation. In agreement with RNA-seq data, 3 splicing forms of IFNL4transcript were detected, including 5, 4 and 3 exons and encoding openreading frames of 179 (SEQ ID NO:2), 131 (SEQ ID NO:5) and 107 (SEQ IDNO:8) amino acids, respectively. FIG. 6 and Table 2 below lists thesequence identifiers assigned to the various open reading frames and therelated nucleic acid molecules, as well as other sequences disclosedherein.

TABLE 2 Sequence Identifiers SEQ ID NO. Organism Description 1 Homop179 DNA encoding sequence (NCBI accession sapiens JN806234) 2 Homoprotein translation of SEQ ID NO: 1 sapiens 3 Homocomplement of SEQ ID NO: 1 sapiens 4 Homop131 DNA encoding sequence (NCBI accession sapiens JN806225) 5 Homoprotein translation of SEQ ID NO: 4 sapiens 6 Homocomplement of SEQ ID NO: 4 sapiens 7 Homop107 DNA encoding sequence (NCBI accession sapiens JN806226) 8 Homoprotein translation of SEQ ID NO: 7 sapiens 9 Homocomplement of SEQ ID NO: 7 sapiens 10 Homo from FIG. 7: CLMDRHDFGsapiens IFNL binding site 1 11 Homo from FIG. 7: DEDLLDKFCTELYQQLNDsapiens IFNL binding site 2 12 Homo from FIG. 7: YFRRITLYLTEKKYSPsapiens IFNL binding site 3 13 Artificial IFNL4- GCCTGCTGCAGAAGCAGAGATSeq/primers p179_forw 14 Artificial IFNL4-p179_rev AGCCGAGCGCAGGACGASeq/primers 15 Artificial IFNL4_VIC AA ATCGCAGAAGGCC Seq/primers(non-risk allele) 16 Artificial IFNL4 FAM-C ATCGCAGCGGCCC Seq/primers(risk allele) 17 Artificial IFNL4_alt_F GCCTGCTGCAGAAGCAGAGATSeq/primers 18 Artificial IFNL4_alt_R GCTCCAGCGAGCGGTAGTG Seq/primers 19Artificial IFNL4_alt_VIC ATCGCAGAAGGCC Seq/primers AA (non-risk allele)20 Artificial IFNL4_alt FAM- ATCGCAGCGGCCC Seq/primers C (risk allele)21 Artificial IFNL4 forw ATGCGGCCGAGTGTCTGGGCC Seq/primers 22 ArtificialIFNL4 rev GAGGCAAGGCCCAGAGTGTGCAG Seq/primers 23 SyntheticAmino acids 44- KALRDRYEEEALSWGQRNCSFRPRRDSPRP peptide 74 of IFNL4- Sp179The first exon of all IFNL4 splicing forms includes a ss469415590-ΔGallele of a previously unreported dinucleotide variant (ss469415590)with two alleles: TT (insertion allele) and ΔG (deletion allele).Protein analysis by BLAST and ClustalW identified strong similaritybetween IFNL4-p179 protein and IFN-λ proteins with IFNL3 (IL28B) beingthe most similar protein, with 29.1% and 40.8% amino acid identity andsimilarity (FIG. 6). The strongest identity between p179 and IFN-λproteins was found within protein sequences corresponding to the IFN-λ Aand F helices, which represent binding sites for the first IFN-λreceptor IFNLR1 (IFN28R1). However, the region of p179 corresponding tothe binding site for the second IFN-λ receptor (IL10R2) was highlydivergent from that found in IFN-λ proteins (FIG. 7). Therefore, thenovel p179 protein is related to but distinct from the interferon-λfamily and other known class-2 cytokines.

Example 4. Production of Recombinant IFNL4 Protein and its Detectionwith an Anti-IFNL4 Monoclonal Antibody

C-terminal His-tag fusion constructs carrying the full-length cDNA formsof IFNL4-p179, p131 and p107 were transduced into a baculoviral strainsf9. Since no expression was detected in cell media, the protein waspurified from cells. High purity (85-90%) was achieved by several roundsof purification and confirmed by Coomassie staining, and Western blotanalyses with anti-His and anti-IFNL4 antibodies (FIG. 8).

Example 5. Development of a Mouse Monoclonal Anti-IFNL4 Antibody

A synthetic peptide KALRDRYEEEALSWGQRNCSFRPRRDSPRPS (SEQ ID NO:23)corresponding to amino acids 44-74 of IFNL4-p179 protein was conjugatedwith KLH (Keyhole Limpet Hemocyanin) for immunization of mice and withbovine serum albumin (BSA) for screening. The post-immunization titerswere accessed from 3 mice using tailbleed serum samples via directenzyme immune assay (EIA) against the BSA-conjugated peptide. One mousewith best IFNL4-specific titer was chosen for monoclonal antibodyproduction. Splenic B cells from the selected animal were isolated andfused with myeloma partner to create IgG hybridomas. Supernatants from35 hybridoma clones were collected and screened for recognition of thepurified recombinant IFNL4 protein, which resulted in isolation of amonoclonal antibody referred to as Mu anti-hu IFNL4 4G1. Hybridomasexpressing Mu anti-hu IFNL4 4G1 were deposited with the American TypeCulture Collection (ATCC®) on Feb. 23, 2012 under ATCC Accession Number:PTA-12575. This example illustrates specific detection of IFNL4 proteinby the anti-IFNL4 monoclonal antibody clone 4G1 by Western blot andconfocal imaging (FIG. 8, 9, 10). Anti-IFNL4 antibody does not recognizeIFN-α, IFNL3 (IL28B) or IFNL4-p107, but is specific for detection ofIFNL4-p179 and, possibly p131 (at least by confocal imaging). Confocalimaging of transiently transfected IFNL4 expression construct inhepatocellular carcinoma HepG2 cells and endogenous IFNL4 expression inprimary human hepatocytes activated with Poly I:C shows intracellularexpression (FIG. 10). IFNL4 was not detected by a negative isotypecontrol—anti-IgG antibody. Similar cellular localization was detectedboth by anti-Halo antibody for the fusion IFNL4-Halo protein and byanti-IFNL4 antibody in HepG2 cells transfected with IFNL4-p179construct. Location of epitopes for mouse and rabbit anti-IFNL4antibodies and specific detection patterns are indicated on FIG. 11.

Example 6. Ability of IFNL4 Protein to Activate the JAK/STAT Pathway

Biological activity of IFNL4 expression construct and IFNL4 recombinantpurified protein, as well as expression constructs for five otherprotein isoforms, was evaluated as the ability to activate reporterconstructs for 45 human signaling pathways in transiently transfectedHepG2 cells. Luciferase Cignal 45-Pathway Finder Reporter Arrays wereused according to instructions (Qiagen, full list of reporters tested isavailable athttp://www.sabiosciences.com/reporter_assay_product/HTML/CCA-901L.html).Cells were transfected with expression constructs for p179, p170, p143,p131, p124 and p107 or treated for 24 h with purified recombinantproteins −10 ng/ml of IFN-α or IFNL3 and/or IFNL4-p179. Of all theconstructs and proteins tested, only transient transfection withexpression construct for IFNL4 and treatment with purified IFN-α (type-Iinterferon) and IFNL3 (type-III interferon) activated theInterferon-Stimulated Response Element (ISRE)-Luc and IRF3-Luc reporters(FIG. 12).

Type I interferons (IFN-α and -β) and type III interferons (IFN-λ)mediate signaling through STAT1 and STAT2 components of theJAK/STAT-signal transduction pathway. The ISRE-Luc Cignal reporter(Qiagen) encodes the firefly luciferase reporter gene under the controlof a minimal (m)CMV promoter linked with tandem repeats which consistsof STAT1/STAT2 binding sites (TAGTTTCACTTTCCC)n that constitute theISRE. The cells were co-transfected with constitutively expressingrenilla luciferase, which serves as an internal control for normalizingtransfection efficiencies and monitoring cell viability. Some cells werealso co-transfected with constructs expressing IFNL4 proteins (p179,p131 and p107) or an empty vector (mock construct), for 72 hours. Othercells were treated with purified recombinant proteins—1, 10 or 100 ng/mlof IFN-α or IFNL3 (IL28B), or IFNL4-p179, p107, for 24 h. All studieswere performed using at least 8 biological replicates. Following thespecified periods of time, the luciferase and renilla expression levelswere measured and luciferase/renilla ratios were calculated in relationto mock-transfected samples.

The results of the analysis for individual ISRe-Luc assay (FIG. 13) showsignificant dose-response signaling by of IFN-α or IFNL3, as well asactivation by transfected IFNL4-p179, but not by transfected p107, p131,which showed below 25% activity of IFNL4-p179 protein. All studies wereperformed using at least 8 biological replicates. A HepG2 cell linestably expressing the same ISRE-Luc reporter construct was generated bytransduction of cells with a Luciferase Cignal Lenti ISRE reporterconstruct (Qiagen) and selection of positive clones by growth inDMEM+10% FBS with 1× Antibiotic-Antimycotic (Life Technologies) and 2ug/mL puromycin. The best HepG2-ISRE-stable clones were identified bytesting with purified recombinant IFN-α and IFNL3. The results detectedby screening of 45 reporters were independently validated by transienttransfections in the HepG2 stable ISRE-Luc cell line (FIG. 13).Transient transfection of IFNL4 construct also induced antiviralactivity in Huh7-Lunet cells carrying a subgenomic HCV replicon linkedwith the luciferase reporter, compared to constructs for non-functionalprotein isoforms p131 or p107 or an empty GFP vector (mock). Theexperiments were performed in a 48-well plate and luciferase expressionwas measured 48 hours post-transfection (FIG. 13).

Example 7. Analysis of STAT1/STAT2 Phosporylation

HepG2 cells in 6-well plates were untreated or transfected withexpression constructs or empty Halo-tag vector, or were treated for 1hour at 37° C. with 50 ng/ml of recombinant IFNL3. Equal amounts (50μg/lane) of whole-cell lysates prepared 48 hours post-transfection wereused for analysis by Western blotting. Detection was performed withrabbit anti-phospho-Tyr701-STAT1 (Cell Signaling Technology) and rabbitanti-phospho-Tyr689-STAT2 (Millipore) antibodies. The blots werestripped and re-probed with rabbit anti-STAT1 and anti-STAT2 antibodies(Santa Cruz Biotechnology) to measure the levels of total STAT1 andSTAT2 proteins. Only treatment with IFNL3 and transient expression withIFNL4 construct induced phosphorylation of STAT1 and STAT2 (FIG. 14).

Example 8. Global Analysis of Transcriptome and Pathway Analysis and theAbility of Intracellularly Expressed IFNL4 Protein to Induce Expressionof Interferon-Stimulated Genes (ISGs)

HepG2 cells were mock-transfected with an empty Halo-tag vector or withIFNL4-Halo expression construct. High-quality RNA (RIN ˜10) preparedfrom transfected cells (48 hours) was used for sequencing with HiSeq2000 (Illumina), generating ˜300M reads per sample. Standard analysisidentified 535 transcripts with >2-fold difference in expression and anFDR<0.05. Ingenuity Pathway Analysis (IPA) performed on this setnominated a list of pathways and specific transcripts (FIG. 15). mRNAexpression of selected transcripts was evaluated in specific conditions,in 4 biological replicates, with pathway-based RT² Profiler PCR arrays,according to instructions (Qiagen)(FIG. 15). HepG2 cells were untreated,or transfected with empty vector (mock); IFNL4-p179, IFNL4-p131 orIFNL4-p107; or treated for 24 hours with 10 ng/ml of IFN-α or IFN-λ,alone of after transfection with mock or IFNL4-p179. Transfected anduntransfected cells were treated with IFN-α or IFN-λ at the same timepoint, corresponding to 24 hours post-transfection and harvested at thesame time. Expression of all transcripts on the array (n=90) wasanalyzed by qRT-PCR pathway profiler arrays with individual specificassays and normalized to expression of four endogenous controls (ACTB,GAPDH, HPRT1 and RPL13A) measured in the same samples. The data ispresented on log 2 scale—less negative values indicate higherexpression. Error bars indicate mean values with 95% confidenceintervals (FIG. 15).

Transient transfection with IFNL4 construct activated the same set ofISGs induced by interferons. In samples already expressing IFNL4 nofurther activation of ISGs could not be achieved by additional treatmentwith interferons.

Example 9. Differential Activation of ISRE-Luc Reporter in Human CellLines

ISRE-Luc reporter was transiently co-transfected into HepG2(hepatocellular carcinoma), 293T (embryonal kidney) and HeLa (cervicalcarcinoma) cell lines, together with empty vector, or constructs forIFNL4, on non-functional forms p131 or 107. Alternatively, ISRE-Lucreporter transfected cells were treated with interferons, at indicatedconcentrations. ISRE-Luc activation by IFNL4 was induced only in HepG2and 293T cells (FIG. 16).

Example 10. Site-Directed Mutagenesis of IFNL4

Further, 83 single-point mutants of IFNL4-p179 were created and theiractivity was evaluated after transient expression in HepG2 cells, usingthe ISRE-Luc reporter and compared to activity of IFNL4-p179 (FIG. 17).Site directed mutagenesis was performed using the QuikChange LightningSite-Directed Mutagenesis Kit (Agilent). Primers were designed to changethe first base of a codon in order to mutate specific IFNL4 amino acids.The original IFNL4-p179 plasmid was used as template for individual PCRreactions with mutagenesis primer pairs. The PCR products were digestedwith DpnI enzyme to eliminate original non-mutated template, the derivedplasmids were pooled and transformed into One Shot TOP10 CompetentCells. Individual colonies were sequenced for validation. FIG. 17 andTable 3 show distribution and type of IFNL4 mutants and their relativepotential to transactivate ISRE-Luc reporter construct.

TABLE 3 Induction of a transiently transfected ISRE-Luc reporterconstruct in HepG2 cells by IFNL4 mutants. Activity of mutants isrelative to IFNL4-p179. t-test to number group residue AA change fold toIFNL4 IFNAN average fold 1 cysteines 17 C 17 Y (SNP) 1.04 6.32E−01 0.282 27 C 27 G 0.09 3.00E−06 3 62 C 62 R 0.26 3.61E−10 4 76 C 76 S 0.067.24E−13 5 122 C 122 G 0.11 3.70E−06 6 152 C 152 G 0.13 4.65E−06 7 178 C178 G 0.25 4.67E−12 8 conserved 19 V 19 M 1.05 6.37E−01 0.66 9 25 R 25 G0.92 3.02E−01 10 34 S 34 P 0.05 2.36E−12 11 35 L 35 M 0.95 4.55E−01 1237 P 37 A 0.11 4.90E−11 13 40 L 40 M 0.77 1.14E−03 14 42 A 42 P 0.081.26E−11 15 44 K 44 E 0.10 2.48E−11 16 48 D 48 H 0.44 4.12E−10 17 51 E51 K 0.45 1.23E−07 18 52 E 52 K 1.14 5.35E−03 19 55 L 55 M 0.99 9.11E−0120 64 F 64 V 0.95 4.71E−01 21 69 D 69 H 0.88 2.47E−02 22 78 R 78 G 0.726.74E−05 23 87 A 87 P 0.15 9.63E−06 24 92 V 92 M 0.90 3.88E−02 25 93 L93 I 1.08 7.23E−02 26 101 L 101 M 1.00 9.77E−01 27 112 L 112 M 0.354.80E−11 28 121 A 121 P 1.09 7.04E−02 29 126 A 126 P 0.84 1.65E−01 30128 P 128 A 0.89 1.69E−01 31 140 R 140 G 0.92 2.26E−01 32 149 S 149 P0.88 1.15E−02 33 150 P 150 A 1.15 7.06E−02 34 155 A 155 P 0.82 1.87E−0335 156 S 156 G 1.12 2.78E−02 36 157 V 157 L 0.48 3.40E−07 37 159 F 159 V0.70 2.44E−05 38 160 N 160 D 0.49 4.57E−08 39 161 L 161 F 0.25 5.21E−0840 163 R 163 G 0.14 8.74E−13 41 164 L 164 M 0.70 1.24E−05 42 165 L 165 F0.24 5.08E−12 43 166 T 166 P 0.40 4.35E−10 44 169 L 169F 0.78 5.92E−0545 173 A 173 P 0.12 6.59E−12 46 non-conserved 2 R 2 G 1.07 3.13E−01 0.9047 3 P 3 A 0.77 7.58E−03 48 4 S 4 G 1.07 2.51E−01 49 5 V 5 F 0.844.52E−02 50 10 A 10 P 1.13 7.06E−02 51 11 A 11 P 0.98 7.69E−01 52 13 L13 M 0.86 4.98E−03 53 15 V 15 F 1.04 5.95E−01 54 20 I 20 V 1.24 5.50E−0455 21 A 21 P 0.99 8.56E−01 56 28 L 28 M 1.15 1.64E−01 57 29 L 29 F 0.242.79E−06 58 30 S 30 P 0.12 1.26E−07 59 32 Y 32 N 0.08 1.41E−12 60 50 Y50 N 0.29 6.52E−11 61 57 W 57 R 1.18 1.42E−01 62 60 R 60 P (SNP) 1.441.79E−01 63 61 N 61 H 1.00 9.43E−01 64 63 S 63 P 1.31 2.24E−04 65 65 R65 G 0.89 2.93E−01 66 70 P 70 S (SNP) 1.10 1.68E−01 67 71 P 71 T 0.997.85E−01 68 72 R 72 G 0.95 3.56E−01 69 102 L 102 F 1.01 9.24E−01 70 111L 111 M 0.26 4.88E−13 71 116 G 116 R 0.98 7.46E−01 72 118 D 118 H 0.473.09E−04 73 120 A 120 P 0.13 4.42E−06 74 129 G 129 A 0.98 9.86E−01 75130 S 130 P 0.81 7.95E−02 76 132 R 132 G 1.08 6.74E−02 77 135 P 135 A0.99 6.86E−01 78 139 K 139 R 1.08 2.00E−01 79 143 K 143 E 1.18 3.24E−0280 146 R 146 K 1.19 5.59E−03 81 154 K 154 E 1.05 4.54E−01 82 158 V 158 I0.91 4.22E−01 83 162 L 162 M 1.25 8.27E−03Analysis of 83 IFNL4 mutants separated in 3 groups: cysteines (n=7),non-cysteine residues conserved (n=38) and non-conserved in IFNL3(n=38). The analysis showed that the 6 non-polymorphic cysteines arecritical for biological activity of IFNL4 as their mutations eliminateIFNL4 biological activity. Only one cysteine, corresponding to a naturalgenetic polymorphism Cys17Tyr, did not affect biological activity ofIFNL4. Mutants of residues conserved between IFNL3 and IFNL4 showstronger impact on biological activity of IFNL4 (FIG. 18).

Example 11. Analysis of Biological Activity of 3 Natural PolymorphicVariants of IFNL4

When IFNL4 is created by ss469415590-ΔG allele, the protein may carry 3additional coding non-synonymous variants (rs73555604 (Cys17Tyr) in exon1, rs142981501 (Arg60Pro) and rs117648444 (Pro70Ser) in exon 2), allpresent in different IFNL4 haplotypes (FIG. 19). Biological activity ofthese variants (Cys17Tyr, Arg60Pro and Pro70Ser) was compared to aconstruct for the wild-type IFNL4 after transient expression of allexpression constructs into HepG2 cells, in three biological replicates.RNA was extracted 48 hours post-transfection, converted to cDNA andassayed for 96 qRT-PCR assays included in the anti-viral qPCR panel(Qiagen). Expression of all transcripts was normalized to expression of4 endogenous controls. Transcripts significantly differing (n=33)between cells transfected by WT-IFNL4 and mutants were used for furtheranalysis with principal components analysis (PCA). Principal componentsanalysis (PCA) based on expression of 33 transcripts involved inantiviral response measured by qRT-PCR in HepG2 transiently transfectedwith specific allelic protein constructs, in three biologicalreplicates. The PCA plot shows that Pro70Ser mutant affects expressionof transcripts differently from both the WT-IFNL4 and the group ofCys17Tyr and Arg60Pro mutants which cluster close to each other. Heatmapplot of transcripts with expression significantly affected by transientexpression of IFNL4 allelic protein constructs (WT-IFNL4, Cys17Tyr,Arg60Pro and Pro70Ser) in HepG2 cells, based on results of experiment onFIG. 20. Mutants Cys17Tyr and Arg60Pro show similar effects on thesetranscripts, while Pro70Ser showed most difference with WT-IFNL4,causing lower expression of IL15, IL18, CTSB, FOS and SPP1 transcriptscompared to cells transfected with WT-IFNL4, and higher expression ofDAK, IRF7, DHX58 and APOBEC3G transcripts compared to cells transfectedwith WT-IFNL4. Color chart corresponds to log 2 scale difference inexpression caused by mutants compared to WT-IFNL4.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed:
 1. An isolated protein that comprises at least about 30 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 2. The isolated protein of claim 1, wherein the protein is capable of activating the JAK/STAT-signal transduction pathway.
 3. The isolated protein of claim 1, wherein the isolated protein comprises at least about 40 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 4. The isolated protein of claim 1, wherein the isolated protein comprises at least about 60 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 5. The isolated protein of claim 1, wherein the isolated protein comprises at least about 80 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 6. The isolated protein of claim 1, wherein the isolated protein comprises at least about 100 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 7. The isolated protein of claim 1, wherein the isolated protein comprises at least about 110 contiguous amino acids from an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
 8. The isolated protein of claim 1, wherein the isolated protein comprises at least about 140 contiguous amino acids from SEQ ID NO:2.
 9. An isolated protein comprising a sequence of at least 50 contiguous amino acids, wherein the at least 50 contiguous amino acid sequence is at least 92% identical over its entire length to an at least 50 contiguous amino acid sequence from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 10. The isolated protein of claim 9, wherein the isolated protein comprises a sequence of at least 150 contiguous amino acids that is at least 92% identical over its entire length to an at least 150 contiguous amino acid sequence from SEQ ID NO:2.
 11. The isolated protein of claim 9, wherein the at least 50 contiguous amino acid sequence comprises at least one sequence feature selected from the group consisting of: a. a cysteine residue at the position corresponding to position 27 of SEQ ID NO:2; b. an leucine residue at the position corresponding to position 29 of SEQ ID NO:2 c. a serine residue at the position corresponding to position 30 of SEQ ID NO:2 d. a tyrosine residue at the position corresponding to position 32 of SEQ ID NO:2 e. a serine residue at the position corresponding to position 34 of SEQ ID NO:2 f. a proline residue at the position corresponding to position 37 of SEQ ID NO:2; g. a leucine residue at the position corresponding to position 40 of SEQ ID NO:2; h. an alanine residue at the position corresponding to position 42 of SEQ ID NO:2; i. a lysine residue at the position corresponding to position 44 of SEQ ID NO:2; j. an aspartic acid residue at the position corresponding to position 48 of SEQ ID NO:2; k. a tyrosine residue at the position corresponding to position 50 of SEQ ID NO:2; l. a leucine residue at the position corresponding to position 111 of SEQ ID NO:2; m. a leucine residue at the position corresponding to position 112 of SEQ ID NO:2; n. an aspartic acid residue at the position corresponding to position 118 of SEQ ID NO:2; o. an alanine residue at the position corresponding to position 120 of SEQ ID NO:2; p. a cysteine residue at the position corresponding to position 122 of SEQ ID NO:2; q. a cysteine residue at the position corresponding to position 152 of SEQ ID NO:2; r. a valine residue at the position corresponding to position 157 of SEQ ID NO:2; s. an asparagine residue at the position corresponding to position 160 of SEQ ID NO:2; t. a leucine residue at the position corresponding to position 161 of SEQ ID NO:2; u. an arginine residue at the position corresponding to position 163 of SEQ ID NO:2 v. a leucine residue at the position corresponding to position 165 of SEQ ID NO:2; w. a threonine residue at the position corresponding to position 166 of SEQ ID NO:2; x. an alanine residue at the position corresponding to position 173 of SEQ ID NO:2; and y. a cysteine residue at the position corresponding to position 178 of SEQ ID NO:2. z.
 12. The isolated protein of claim 9, wherein the isolated protein comprises an amino acid sequence at least 92% identical over its entire length to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 13. The isolated protein of claim 1, wherein the isolated protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
 14. The isolated protein of claim 1, wherein the protein possess at least one activity selected form the group consisting of eliciting an antibody that selectively binds a protein consisting of SEQ ID NO:2, selectively binding a compound that binds to a protein consisting of SEQ ID NO:2, activating expression of the JAK/STAT pathway, and inducing expression of at least one ISG listed in FIG.
 15. 15. The isolated protein of claim 1, wherein the protein activates the JAK/STAT pathway.
 16. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a. a nucleic acid sequence encoding the isolated protein of claim 1; and b. a nucleic acid sequence fully complementary to the nucleic acid sequence of (a).
 17. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9.
 18. A plasmid comprising the nucleic acid molecule of claim
 16. 19. A virus comprising the nucleic acid molecule of claim
 16. 20. An isolated antibody that inhibits the binding of an antibody that selectively binds to the isolated protein of claim
 9. 21. An isolated antibody that selectively binds to the isolated protein of claim
 9. 22. An variant IFNL4 polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to IFNL4-p179, p131 and p107 (SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8) wherein the variant polypeptide: (a) has at least one amino acid substitution, as compared to SEQ ID NO:2, selected from A10P, A11P, L13M, V15F, C17Y, V19M, I20V, A21P, R26G, L28M, L35M, L40M, E52K, L55M, W57R, R60P, N61H, S63P, F64V, R65G, D69H, P70S, P71T, R72G, R78G, V92M, L931, L101M, L102F, G116R, A121P, A126P, P128A, G129A, S130P, R132G, P135A, K139R, R140G, K143E, R146K, S149P, P150A, K154E, A155P, S156G, V1581, F159V, L162M, L164M, and L169F.; (b) has at least one amino acid substitution as compared to SEQ ID NO:2 selected from A10P, A11P, L13M, V15F, C17Y, V19M, I20V, A21P, R26G, L28M, L35M, L40M, E52K, L55M, W57R, R60P, N61H, S63P, F64V, R65G, D69H, P70S, P71T, R72G, G116R, A121P, A126P, P128A, G129A, S130P, R132G, P135A, K139R, R140G, K143E, R146K, S149P, P150A, K154E, A155P, S156G, V1581, F159V, L162M, L164M, and L169F; (c) has at least one amino acid substitution as compared to SEQ ID NO:2 selected from A10P, A11P, L13M, V15F, C17Y, V19M, I20V, A21P, R26G, L28M, L35M, L40M, G116R, A121P, A126P, P128A, G129A, S130P, R132G, P135A, K139R, R140G, K143E, R146K, S149P, P150A, K154E, A155P, S156G, V1581, F159V, L162M, L164M, and L169F; or (d) has at least one amino acid substitution as compared to SEQ ID NO:2 selected from C27G, S34P, P37A, D48H, C62R, A87P, D118H, A120P, C122G, C152G, V157L, N160D, L161F, L164M, L165F, T166P, L169F, and A173P.
 23. A method for predicting the likelihood of an individual to spontaneously clear an HCV infection, the method comprising: a) obtaining a biological sample from the individual; and, b) analyzing the sample to determine the presence or absence of an IFNL4 mRNA or protein of the present invention; wherein the presence of an IFNL4 protein of the present invention indicates the likelihood of the individual spontaneously clearing an HCV infection.
 24. The method of claim 23, wherein the absence of IFNL4 mRNA or protein indicates the individual is predicted to spontaneously clear an HCV infection.
 25. The method of claim 23, wherein the presence of IFNL4 mRNA or protein indicates the individual is predicted to be unable to spontaneously clear an HCV infection.
 26. A method for predicting the likelihood that an individual will respond to a treatment for HCV infection, the method comprising: a) obtaining a biological sample from the individual; b) analyzing the sample to determine the presence or absence of an IFNL4 protein of the present invention; wherein the presence of an IFNL4 protein of the present invention indicates the likelihood the individual will respond to treatment for an HCV infection.
 27. The method of claim 26, wherein the absence of IFNL4 protein indicates the individual is predicted to respond to treatment for an HCV infection.
 28. The method of claim 26, wherein the presence of IFNL4 protein indicates the individual is predicted to be unable to respond to treatment for an HCV infection.
 29. A method for predicting the likelihood of an individual to spontaneously clear an HCV infection, the method comprising: a) obtaining a biological sample from an individual; and, b) determining the level of IFNL4 protein present in the sample, if any; wherein the level of IFNL4 protein present in the sample indicates the likelihood the individual will spontaneously clear an HCV infection.
 30. The method of claim 29, wherein a level of IFNL4 in the sample less than the level of IFNL4 protein present in a subject known to be able to clear an HCV infection indicates the individual is predicted to be able to clear an HCV infection.
 31. The method of claim 29, wherein a level of IFNL4 in the sample greater than the level of IFNL4 protein present in a subject known to be unable to clear an HCV infection indicates the individual is predicted to be unable to clear an HCV infection.
 32. A method for treating a patient suffering from a chronic hepatitis C virus infection, the method comprising: a) obtaining a biological sample from the individual; b) analyzing the sample to determine the presence, absence or level of IFNL4 protein present in the sample; and, c) determining whether or not to administer treatment based on the presence, absence or amount of IFNL4 protein present in the sample.
 33. A method for identifying regulators of IFNL4 protein activity, the method comprising: a) incubating IFNL4 protein under conditions that allow measurement of IFNL4 protein activity; b) measuring the resultant IFNL4 activity; c) incubating IFNL4 protein in the presence of a test compound under the conditions used in step (a); d) measuring the resultant IFNL4 activity; and e) comparing the IFNL4 activity obtained in step (b) with the IFNL4 activity obtained in step (d); wherein if the difference between the IFNL4 activity obtained in step (b) and the IFNL4 activity obtained in step (d) is statistically significant, identifying the test compound as a regulator of IFNL4 activity.
 34. The method of claim 23, 26 or 29, wherein if IFNL4 is detected, the method further comprises analyzing the detected protein for the presence of one or more mutations.
 35. The method of claim 34, wherein the mutations are selected from the group consisting of Cys17Tyr, Arg60Pro and Pro70Ser.
 36. A kit for determining the presence, absence or level of IFNL4 protein in a sample, the kit comprising the antibody of claim
 21. 37. The kit of claim 36, wherein the kit comprises instructions for determining the ability of an individual to spontaneously clear an HCV infection.
 38. The kit of claim 36, wherein the kit comprises instructions for determining the ability of an individual to respond to treatment for an HCV infection. 